module 2: ultra structure of cell
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This document provides an overview of cell organelle structure and function. It compares prokaryotic and eukaryotic cells, describing key differences such as nucleus, organelles, and cell size. Major eukaryotic organelles like the nucleus, mitochondria, chloroplasts, and endoplasmic reticulum are summarized in terms of their main functions and structures. The document also discusses the structure and functions of specific organelles like the nucleus, mitochondria, and chromosomes in more detail.
![Ultra structure of cell,
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the way to lysosomes for degradation, or they can be recycled back to the plasma membrane.
Molecules are also transported to endosomes from the trans-Golgi network and either continue to
lysosomes or recycle back to the Golgi. Endosomes can be classified as early, sorting, or late
depending on their stage post internalizationEndosomes represent a major sorting compartment of
the endomembrane system in cells. In HeLa cells, endosomes are approximately 500 nm in diameter
when fully mature.
Function
Endosomes provide an environment for material to be sorted before it reaches the degradative
lysosome For example, LDLis taken into the cell by binding to the LDL receptor at the cell surface.
Upon reaching early endosomes, the LDL dissociates from the receptor, and the receptor can be
recycled to the cell surface. The LDL remains in the endosome and is delivered to lysosomes for
processing. LDL dissociates because of the slightly acidified environment of the early endosome,
generated by a vacuolar membrane proton pump V-ATPase. On the other hand, EGF and the EGF
receptor have a pH-resistant bond that persists until it is delivered to lysosomes for their
degradation. The mannose 6-phosphate receptor carries ligands from the Golgi destined for the
lysosome by a similar mechanism.
Types
Endosomes comprise three different compartments: early endosomes, late endosomes, and recycling
endosomes. They are distinguished by the time it takes for endocytosed material to reach them, and
by markers such as rabs They also have different morphology. Once endocytic vesicles have uncoated,
they fuse with early endosomes. Early endosomes then mature into late endosomes before fusing with
lysosomes.
Early endosomes mature in several ways to form late endosomes. They become increasingly acidic
mainly through the activity of the V-ATPase. Many molecules that are recycled are removed by
concentration in the tubular regions of early endosomes. Loss of these tubules to recycling pathways
means that late endosomes mostly lack tubules. They also increase in size due to the homotypic fusion
of early endosomes into larger vesicles. Molecules are also sorted into smaller vesicles that bud from
the perimeter membrane into the endosome lumen, forming lumenal vesicles; this leads to the
multivesicular appearance of late endosomes and so they are also known as multivesicular
bodies (MVBs). Removal of recycling molecules such as transferrin receptors and mannose 6-
phosphate receptors continues during this period, probably via budding of vesicles out of
endosomes.] Finally, the endosomes lose RAB5A and acquire RAB7A, making them competent for
fusion with lysosomesFusion of late endosomes with lysosomes has been shown to result in the
formation of a 'hybrid' compartment, with characteristics intermediate of the two source
compartments.
For example, lysosomes are more dense than late endosomes, and the hybrids have an intermediate
density. Lysosomes reform by recondensation to their normal, higher density. However, before this
happens, more late endosomes may fuse with the hybrid.
Some material recycles to the plasma membrane directly from early endosomes but most traffics via
recycling endosomes.](https://image.slidesharecdn.com/unit1-ultrastructureofcell-170825115031/85/module-2-ultra-structure-of-cell-18-320.jpg)
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Early endosomes consist of a dynamic tubular-vesicular network (vesicles up to 1 µm in
diameter with connected tubules of approx. 50 nm diameter). Markers include RAB5Aand
RAB4, Transferrin and its receptor and EEA1.
Late endosomes, also known as MVBs, are mainly spherical, lack tubules, and contain many
close-packed lumenal vesicles. Markers include RAB7, RAB9, and mannose 6-phosphate
receptors.[11]
Recycling endosomes are concentrated at the microtubule organizing center and consist of a
mainly tubular network. Marker; RAB11.
More subtypes exist in specialized cells such as polarized cells and macrophages.
Phagosomes, macropinosomes and autophagosomes mature in a manner similar to endosomes, and
may require fusion with normal endosomes for their maturation. Some intracellular pathogens
subvert this process, for example, by preventing RAB7 acquisition. Late endosomes/MVBs are
sometimes called endocytic carrier vesicles, but this term was used to describe vesicles that bud from
early endosomes and fuse with late endosomes. However, several observations (described above)
have now demonstrated that it is more likely that transport between these two compartments occurs
by a maturation process, rather than vesicle transport.
Another unique identifying feature that differs between the various classes of endosomes is the lipid
composition in their membranes. Phosphotidyl inositol phosphates (PIPs), one of the most
important lipid signaling molecules, is found to differ as the endosomes mature from early to
late. PI(4,5)P2 is present on plasma membranes, PI(3)P on early endosomes, PI(3,5)P2 on late
endosomes and PI(4)P on the trans Golgi network. These lipids on the surface of the endosomes help
in the specific recruitment of proteins from the cytosol, thus providing them an identity. The inter-
conversion of these lipids is a result of the concerted action of
phosphoinositide kinases and phosphatases that are strategically localized
Pathways
Diagram of the pathways that intersect endosomes in the endocytic pathway of animal cells.
Examples of molecules that follow some of the pathways are shown, including receptors for EGF,
transferrin, and lysosomal hydrolases. Recycling endosomes, and compartments and pathways found
in more specialized cells, are not shown.
There are three main compartments that have pathways that connect with endosomes. More
pathways exist in specialized cells, such as melanocytes and polarized cells. For example,
in epithelial cells, a special process called transcytosis allows some materials to enter one side of a
cell and exit from the opposite side. Also, in some circumstances, late endosomes/MVBs fuse with the
plasma membrane instead of with lysosomes, releasing the lumenal vesicles, now called exosomes,
into the extracellular medium.
It should be noted that there is no consensus as to the exact nature of these pathways, and the
sequential route taken by any given cargo in any given situation will tend to be a matter of debate.
Golgi to/from endosomes](https://image.slidesharecdn.com/unit1-ultrastructureofcell-170825115031/85/module-2-ultra-structure-of-cell-19-320.jpg)
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Vesicles pass between the Golgi and endosomes in both directions. The GGAs and AP-1 clathrin-coated
vesicle adaptors make vesicles at the Golgi that carry molecules to endosomes. In the opposite
direction, retromer generates vesicles at early endosomes that carry molecules back to the Golgi.
Some studies describe a retrograde traffic pathway from late endosomes to the Golgi that is mediated
by Rab9 and TIP47, but other studies dispute these findings. Molecules that follow these pathways
include the mannose-6-phosphate receptors that carry lysosomal hydrolases to the endocytic
pathway. The hydrolases are released in the acidic environment of endosomes, and the receptor is
retrieved to the Golgi by retromer and Rab9.
Plasma membrane to/from early endosomes (via recycling endosomes)
Molecules are delivered from the plasma membrane to early endosomes in endocytic vesicles.
Molecules can be internalized via receptor-mediated endocytosis in clathrin-coated vesicles. Other
types of vesicles also form at the plasma membrane for this pathway, including ones
utilising caveolin. Vesicles also transport molecules directly back to the plasma membrane, but many
molecules are transported in vesicles that first fuse with recycling endosomes.[18] Molecules following
this recycling pathway are concentrated in the tubules of early endosomes. Molecules that follow
these pathways include the receptors for LDL, the growth factor EGF, and the iron transport protein
transferrin. Internalization of these receptors from the plasma membrane occurs by receptor-
mediated endocytosis. LDL is released in endosomes because of the lower pH, and the receptor is
recycled to the cell surface. Cholesterol is carried in the blood primarily by (LDL), and transport by
the LDL receptor is the main mechanism by which cholesterol is taken up by cells. EGFRs are
activated when EGF binds. The activated receptors stimulate their own internalization and
degradation in lysosomes. EGF remains bound to the EGFR once it is endocytosed to endosomes. The](https://image.slidesharecdn.com/unit1-ultrastructureofcell-170825115031/85/module-2-ultra-structure-of-cell-20-320.jpg)
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The cytoskeleton can also actively contract, thereby deforming the cell and the cell's
environment and allowing cells to migrate. Moreover, it is involved in many cell
signaling pathways, in the uptake of extracellular material (endocytosis), segregates
chromosomes during cellular division, is involved in cytokinesis (the division of a mother cell
into two daughter cells), provides a scaffold to organize the contents of the cell in space and
for intracellular transport (for example, the movement of vesicles and organelles within the
cell); and can be a template for the construction of a cell wall. Furthermore, it forms
specialized structures, such as flagella, cilia, lamellipodia and podosomes.
A large-scale example of an action performed by the cytoskeleton is muscle contraction.
During contraction of a muscle, within each muscle cell, myosin molecular motors collectively
exert forces on parallel actin filaments. This action contracts the muscle cell, and through the
synchronous process in many muscle cells, the entire muscle.
Microfilaments:
Microfilaments, also called actin filaments, are filamentous structures in
the cytoplasm of eukaryotic cells and form part of the cytoskeleton.
They are primarily composed of polymers of actin, but in cells are modified by and interact
with numerous other proteins.
Microfilament functions include cytokinesis, amoeboid movement and cell motility in general,
changes in cell shape, endocytosis and exocytosis, cell contractility and mechanical stability.
Microfilaments are flexible and relatively strong, resisting buckling by multi-piconewton
compressive forces and filament fracture by nanonewton tensile forces. In inducing cell
motility, one end of the actin filament elongates while the other end contracts, presumably
by myosin II molecular motors.[1] Additionally, they function as part of actomyosin-driven
contractile molecular motors, wherein the thin filaments serve as tensile platforms for
myosin's ATP-dependent pulling action in muscle contraction and pseudopod advancement.
Microfilaments have a tough, flexible framework which helps the cell in movement.
Actin:
Actin can hydrolyze its bound ATP to ADP + Pi, releasing Pi. The actin monomer
can exchange bound ADP for ATP. The conformation of actin is different, depending on whether
there is ATP or ADP in the nucleotide-binding site.](https://image.slidesharecdn.com/unit1-ultrastructureofcell-170825115031/85/module-2-ultra-structure-of-cell-22-320.jpg)
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is protein complex of the inner mitochondrial membrane [14]. It is involved in the translocation of
protons and catalysis of oxygen to water required for ATP synthesis. Most of the COX subunits can be
good mitochondrial markers.
Apoptosis-inducing factor (AIF, PDCD8)
is a 67 kDa protein of the inter-membrane space and is ubiquitously expressed. While in
mitochondria, AIF functions as an oxidoreductase and has anti-apoptotic activity. However, during
apoptotic signals, AIF is released to the cytoplasm and translocates to the nucleus to induce nuclear
apoptosis.
Hexokinase
is a 100 kDa kinase of the outer mitochondrial membrane catalyzing the first step of glycolysis.
Hexokinase phosphorylates hexoses (a six-carbon sugar) to form hexose phosphates (e.g. glucose to
glucose-6-phosphate).
VDAC1
(outer mitochondrial membrane protein porin 1) is the outer mitochondrial membrane receptor for
hexokinase and BCL2L1.
Nuclear markers
There are several proteins used to distinguish the distinct nuclear structures from each other:
Chromatin:
The DNA molecule is condensed as it is wrapped around the four core histones (H2A, H2B, H3,
H4 - which form an octamer) and form the nucleosome. Histone H1 is a protein linker which binds to
distant chromatin areas and compacts it further. The DNA with its histones and other proteins that
associated with it (to regulate transcription, replication, DNA repair etc) is called chromatin.
Therefore, any dye that binds DNA can be used as a chromatin marker. For example, 4',6-
diamidino-2-phenylindole (DAPI) and the dyes Hoechst 33258 and Hoechst 33342 are the most
common. Bromodeoxyuridine (BrdU) is a synthetic nucleoside used to detect proliferating cells. 5-
ethynyl-2'-deoxyuridine (EdU) staining is equally effective.
All core histones are required for nucleosome formation. Therefore, anti-histone antibodies
would mark mainly chromatin. Histone modification specific antibodies can distinguish between
euchromatin (e.g. H3K4me3 or H3K36me3) and heterochromatin (e.g. H4K20me3). Phosphohistone
H3 can also be used to indicate the mitotic state of cells.
Telomeric repeat-binding factor 2-interacting protein 1 (TERF-1, RAP1) is localized at
telomeres and regulates telomere length.
Nuclear envelope:
The nuclear envelope is the lipid bilayer membranous structure surrounding the nucleus and
separates it from the cytoplasm. The inner membrane is comprised of a network of intermediate
filaments, the lamina, while the outer membrane is physically linked to the endoplasmic reticulum,
thus sharing some common proteins. Small holes on the nuclear envelope constitute the nuclear pore
complexes of about 100 nm diameter and they connect the inner with the outer nuclear membrane as
well as import or export proteins to and from the nucleus.](https://image.slidesharecdn.com/unit1-ultrastructureofcell-170825115031/85/module-2-ultra-structure-of-cell-33-320.jpg)
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The microtubule-associated protein 1 light chain 3 (LC3) is also localized at the membrane but when
fully formed it is found at the isolation membranes as well. LC3 can be found on lysosomes but with
less abundance. Caution has been raised as whether Western blot of LC3 and its associated protein
sequestosome 1 (SQSTM1, also known as p62) can truly reflect the status of autophagosomes,
especially in the case of p62.
Lysosome Associated Membrane Protein 1 and 2 (LAMP1; LAMP2) are components of the
lysosomal membrane and therefore constitute excellent lysosomal markers. They have been recently
used to confirm that transmembrane 7 superfamily member 1 (TM7SF1) is a protein of the integral
membrane of the lysosome.
Beta-galactosidase is one of the glycosidases that can be used as lysosomal markers.
Cation-dependent mannose-6-phosphate receptor (M6PR) is involved in the transport of
lysosomal enzymes from the Golgi and cell surface to the lysosomes.
Melanosomal markers
Melanosomes are organelles of melanocytes, skin and retina epithelial cells. Melanosomes contain the
pigment melanin which protects cells from harmful ultraviolet (UV) radiation.
Markers for melanosomes can be tyrosinase, Tyrp1, Dct, OA1, gp100, and MART1 [20].
Peroxisomal markers
Peroxisomes are structures which are housing oxidative reactions, such as fatty acid β-oxidation, and
protect from peroxides.
Fox2p and Fox5p are peroxisomal membrane receptors.
Catalase is a peroxisomal protein which protects cells from the toxic effects of hydrogen peroxide.
Acyl-coenzyme A thioesterase 8 belongs to a group of enzymes that catalyze the hydrolysis of acyl-
CoAs to the free fatty acid and coenzyme A (CoASH), providing the potential to regulate intracellular
levels of acyl-CoAs, free fatty acids and CoASH.
Peroxins (a class of 24 genes) are integral parts of peroxisome development.
Ribosomal markers
Ribosomes are molecular complexes of proteins and RNA molecules (ribonucleoprotein) in which
proteins are synthesized. They are comprised of a small 40S subunit and a large 60S subunit. Several
ribosome-specific proteins can be used as markers.
Antibodies against ribosomal proteins L7a, L26 (component of the 60 S subunit), S3, S6, S10, S11 (40S
subunit) are characteristic examples.
Proteasomal markers
Proteasomes are multi-protein complexes and their function is to degrade proteins by proteolysis.
Each proteasome consists of four stacked rings forming a central pore, the core. A seven-protein
complex (β subunits) of proteolytic enzymes forms the two rings in the interior while seven α subunits
form the entrance through which proteins enter and reach the core.
The proteasome subunits 20S proteasome, 26S proteasome, α7 and Rpn2, Pre6, Cim5 and Scl1 are
commonly used as proteasomal markers.
Cell cycle markers
Mitosis markers](https://image.slidesharecdn.com/unit1-ultrastructureofcell-170825115031/85/module-2-ultra-structure-of-cell-36-320.jpg)
module 2: ultra structure of cell
- 1. LECTURE NOTES IN CELL BIOLOGY Module 2: structure and function of cell organelles AUGUST 24, 2017 SARDAR HUSSAIN Assistant Professor Biotechnology, GSC, cta, sardar1109@gmail.com
- 2. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 1 2016-17 Ultra-structure of cell: Comparison of features of prokaryotic and eukaryotic cells Prokaryotes Eukaryotes Typical organisms bacteria, archaea protists, fungi, plants, animals Typical size ~ 1–5 µm ~ 10–100 µm Type of nucleus nucleoid region; no true nucleus true nucleus with double membrane DNA circular (usually) linear molecules (chromosomes) with histone proteins RNA/protein synthesis coupled in the cytoplasm RNA synthesis in the nucleus protein synthesis in the cytoplasm Ribosomes 50S and 30S 60S and 40S Cytoplasmic structure very few structures highly structured by endomembranes and a cytoskeleton Cell movement flagella made of flagellin flagella and cilia containing microtubules; lamellipodia and filopodia containing actin Mitochondria none one to several thousand Chloroplasts none in algae and plants Organization usually single cells single cells, colonies, higher multi-cellular organisms with specialized cells Cell division binary fission (simple division) mitosis(fission or budding) meiosis Chromosomes single chromosome more than one chromosome Membranes cell membrane Cell membrane and membrane-bound organelles Major eukaryotic organelles Organelle Main function Structure Organisms Notes chloroplast (plastid) photosynthesis, traps energy from sunlight double-membrane compartment plants, protists (rare kleptoplastic organisms) has own DNA; theorized to be engulfed by the ancestral eukaryotic cell (endosymbiosis) endoplasmic reticulum translation and folding of new proteins (rough single-membrane compartment all eukaryotes rough endoplasmic reticulum is covered with ribosomes, has
- 3. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 2 2016-17 endoplasmic reticulum), expression of lipids (smooth endoplasmic reticulum) folds that are flat sacs; smooth endoplasmic reticulum has folds that are tubular Flagellum locomotion, sensory some eukaryotes Golgi apparatus sorting, packaging, processing and modification of proteins single-membrane compartment all eukaryotes cis-face (convex) nearest to rough endoplasmic reticulum; trans-face (concave) farthest from rough endoplasmic reticulum mitochondria energy production from the oxidation of glucose substances and the release of adenosine triphosphate double-membrane compartment most eukaryotes has own DNA; theorized to be engulfed by an ancestral eukaryotic cell (endosymbiosis) vacuole storage, transportation, helps maintain homeostasis single-membrane compartment eukaryotes nucleus DNA maintenance, controls all activities of the cell, RNA transcription double-membrane compartment all eukaryotes contains bulk of genome Minor eukaryotic organelles and cell components Organelle/ Macromolecule Main function Structure Organisms acrosome helps spermatozoa fuse with ovum single-membrane compartment many animals autophagosome vesicle that sequesters cytoplasmic material and organelles for degradation double-membrane compartment all eukaryotes
- 4. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 3 2016-17 centriole anchor for cytoskeleton, organizes cell division by forming spindle fibers Microtubule protein Animals cilium Movement in or of external medium; "critical developmental signaling pathway". Microtubule protein animals, protists, few plants eyespot apparatus detects light, allowing phototaxis to take place green algae and other unicellular photosynthetic organisms such as euglenids glycosome carries out glycolysis single-membrane compartment Some protozoa, such as Trypanosomes. glyoxysome conversion of fat into sugars single-membrane compartment Plants hydrogenosome energy & hydrogen production double-membrane compartment a few unicellular eukaryotes lysosome breakdown of large molecules (e.g., proteins + polysaccharides) single-membrane compartment most eukaryotes melanosome pigment storage single-membrane compartment Animals mitosome probably plays a role in Iron- sulfur cluster (Fe-S) assembly double-membrane compartment a few unicellular eukaryotes that lack mitochondria myofibril myocyte contraction bundled filaments Animals nematocyst stinging coiled hollow tubule Cnidarians nucleolus pre-ribosome production protein-DNA-RNA most eukaryotes parenthesome not characterized not characterized Fungi peroxisome breakdown of metabolic hydrogen peroxide single-membrane compartment all eukaryotes proteasome degradation of unneeded or damaged proteins by proteolysis very large protein complex All eukaryotes, all archaea, some bacteria ribosome (80S) translation of RNA into proteins RNA-protein all eukaryotes vesicle material transport single-membrane compartment all eukaryotes Stress granule mRNA storage membraneless (mRNP complexes) Most eukaryotes
- 5. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 4 2016-17 Prokaryotic organelles and cell components Organelle/ Macromolecule Main function Structure Organisms carboxysome carbon fixation protein-shell compartment some bacteria chlorosome photosynthesis light harvesting complex green sulfur bacteria flagellum movement in external medium protein filament some prokaryotes and eukaryotes magnetosome magnetic orientation inorganic crystal, lipid membrane magnetotactic bacteria nucleoid DNA maintenance, transcription to RNA DNA-protein Prokaryotes plasmid DNA exchange circular DNA some bacteria ribosome (70S) translation of RNA into proteins RNA-protein bacteria and archaea thylakoid photosynthesis photosystem proteins and pigments mostly cyanobacteria mesosomes functions of Golgi bodies, centrioles, etc. small irregular shaped organelle containing ribosomes present in most prokaryotic cells Pilus Adhesion to other cells for conjugation or to a solid substrate to create motile forces. a hair-like appendage sticking out (though partially embedded into) the plasma membrane prokaryotic cells
- 6. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 5 2016-17 Nucleus: structure and function: The nucleus is the largest cellular organelle in animal cells. In mammalian cells, the average diameter of the nucleus is approximately 6 micrometres (µm), which occupies about 10% of the total cell volume. The viscous liquid within it is called nucleoplasm, and is similar in composition to the cytosol found outside the nucleus. It appears as a dense, roughly spherical or irregular organelle. The nuclear envelope, otherwise known as nuclear membrane, consists of two cellular membranes, an inner and an outer membrane, arranged parallel to one another and separated by 10 to 50 nanometres (nm). The nuclear envelope completely encloses the nucleus and separates the cell's genetic material from the surrounding cytoplasm, serving as a barrier to prevent macromolecules from diffusing freely between the nucleoplasm and the cytoplasm. The outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum(RER), and is similarly studded with ribosomes. The space between the membranes is called the perinuclear space and is continuous with the RER lumen. Nuclear pores, which provide aqueous channels through the envelope, are composed of multiple proteins, collectively referred to as nucleoporins. Most proteins, ribosomal subunits, and some DNAs are transported through the pore complexes in a process mediated by a family of transport factors known as karyopherins. Those karyopherins that mediate movement into the nucleus are also called importins, whereas those that mediate movement out of the nucleus are called exportins. In animal cells, two networks of intermediate filaments provide the nucleus with mechanical support: The nuclear lamina forms an organized meshwork on the internal face of the envelope, while less organized support is provided on the cytosolic face of the envelope. Both systems provide structural support for the nuclear envelope and anchoring sites for chromosomes and nuclear pores. The nucleolus is a discrete densely stained structure found in the nucleus. It is not surrounded by a membrane, and is sometimes called a sub organelle. It forms around tandem repeats of rDNA, DNA coding for ribosomal RNA (rRNA). These regions are called nucleolar organizer regions (NOR). The main roles of the nucleolus are to synthesize rRNA and assemble ribosomes. The structural cohesion of the nucleolus depends on its activity, as ribosomal assembly in the nucleolus results in the transient association of nucleolar components, facilitating further ribosomal assembly, and hence further association. When observed under the electron microscope, the nucleolus can be seen to consist of three distinguishable regions: the innermost fibrillar centers (FCs), surrounded by the dense fibrillar component (DFC), which in turn is bordered by the granular component (GC). Transcription of the rDNA occurs either in the FC or at the FC-DFC boundary, and, therefore, when rDNA transcription in the cell is increased, more FCs are detected. Most of the cleavage
- 7. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 6 2016-17 and modification of rRNAs occurs in the DFC, while the latter steps involving protein assembly onto the ribosomal subunits occur in the GC Functions of the Nucleus It controls the heredity characteristics of an organism. It is responsible for protein synthesis, cell division, growth and differentiation. Stores heredity material in the form of deoxy-ribonucleic acid (DNA) strands. Also stores proteins and ribonucleic acid (RNA) in the nucleolus. It is a site for transcription process in which messenger RNA (m RNA) are produced for protein synthesis. Aids in exchange of DNA and RNA (heredity materials) between the nucleus and the rest of the cell. Nucleolus produces ribosomes and is known as protein factories. It also regulates the integrity of genes and gene expression. Chromosomes: The cell nucleus contains the majority of the cell's genetic material in the form of multiple linear DNA molecules organized into structures called chromosomes. Each human cell contains roughly two meters of DNA. During most of the cell cycle these are organized in a DNA-protein complex known as chromatin, and during cell division the chromatin can be seen to form the well-defined chromosomes familiar from a karyotype. A small fraction of the cell's genes are located instead in the mitochondria. There are two types of chromatin. Euchromatin is the less compact DNA form, and contains genes that are frequently expressed by the cell. The other type, heterochromatin, is the more compact form, and contains DNA that is infrequently transcribed. This structure is further categorized into facultative heterochromatin, consisting of genes that are organized as heterochromatin only in certain cell types or at certain stages of development, and constitutive heterochromatin that consists of chromosome structural components such as telomeres and centromeres. During interphase the chromatin organizes itself into discrete individual patches, called chromosome territories. Antibodies to certain types of chromatin organization, in particular, nucleosomes, have been associated with a number of autoimmune diseases, such as systemic lupus erythematosus. These are known as anti-nuclear antibodies (ANA) and have also been observed in concert with multiple sclerosis as part of general immune system dysfunction.
- 8. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 7 2016-17 Mitochondria: Structure and Function: A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which increase its surface area. A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes have different properties. Because of this double-membraned organization, there are five distinct parts to a mitochondrion. They are: 1. the outer mitochondrial membrane, 2. the intermembrane space (the space between the outer and inner membranes), 3. the inner mitochondrial membrane, 4. the cristae space (formed by infoldings of the inner membrane), and 5. the matrix (space within the inner membrane). Mitochondria stripped of their outer membrane are called mitoplasts. Outer membrane It contains large numbers of integral membrane proteins called porins. These porins form channels. The outer membrane also contains enzymes involved in such diverse activities as the elongation of fatty acids, oxidation of epinephrine, and the degradation of tryptophan. These enzymes include monoamine oxidase, rotenone- insensitive NADH-cytochrome c- reductase, kynurenine hydroxylase and fatty acid Co-A ligase. The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is
- 9. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 8 2016-17 important in the ER-mitochondria calcium signaling and is involved in the transfer of lipids between the ER and mitochondria. Outside the outer membrane there are small (diameter: 60Å) particles named sub-units of Parson. Intermembrane space The intermembrane space is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules, such as ions and sugars, in the intermembrane space is the same as in the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c. Inner membrane The inner mitochondrial membrane contains proteins with five types of functions: 1. Those that perform the redox reactions of oxidative phosphorylation 2. ATP synthase, which generates ATP in the matrix 3. Specific transport proteins that regulate metabolite passage into and out of the matrix 4. Protein import machinery 5. Mitochondrial fusion and fission protein In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. Cristae The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. Matrix The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle. Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins.
- 10. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 9 2016-17 Mitochondria play a central role in many other metabolic tasks, such as: Signaling through mitochondrial reactive oxygen species Regulation of the membrane potential Apoptosis-programmed cell death Calcium signaling (including calcium-evoked apoptosis) Regulation of cellular metabolism Certain heme synthesis reactions Steroid synthesis. Hormonal signaling Chloroplast: The chloroplasts are double membrane bound organelles and are the site of photosynthesis. The chloroplasts have a system of three membranes: the outer membrane, the inner membrane and the thylakoid system. The outer and the inner membrane of the chloroplast enclose a semi-gel-like fluid known as the stroma. This stroma makes up much of the volume of the chloroplast, the thylakoids system floats in the stroma. Outer membrane - It is a semi-porous membrane and is permeable to small molecules and ions, which diffuses easily. The outer membrane is not permeable to larger proteins. Intermembrane Space - It is usually a thin intermembrane space about 10-20 nanometers and it is present between the outer and the inner membrane of the chloroplast. Inner membrane - The inner membrane of the chloroplast forms a border to the stroma. It regulates passage of materials in and out of the chloroplast. In addition of regulation activity, the fatty acids, lipids and carotenoids are synthesized in the inner chloroplast membrane.
- 11. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 10 2016-17 Stroma Stroma is a alkaline, aqueous fluid which is protein rich and is present within the inner membrane of the chloroplast. The space outside the thylakoid space is called the stroma. The chloroplast DNA chlroplast ribosomes and the thylakoid sytem, starch granules and many proteins are found floating around the stroma. Thylakoid System The thylakoids are arranged in stacks known as grana. Each granum contains around 10-20 thylakoids. Thylakoids are interconnected small sacks, the membranes of these thylakoids is the site for the light reactions of the photosynthesis to take place. Important protein complexes which carry out light reaction of photosynthesis are embedded in the membranes of the thylakoids. The Photosystem I and the Photosystem II are complexes that harvest light with chlorophyll and carotenoids, they absorb the light energy and use it to energize the electrons. Functions of chloroplast: In plants all the cells participate in plant immune response as they lack specialized immune cells. The chloroplasts with the nucleus and cell membrane and ER are the key organelles of pathogen defense. The most important function of chloroplast is to make food by the process of photosynthesis. Food is prepared in the form of sugars. During the process of photosynthesis sugar and oxygen are made using light energy, water, and carbon dioxide. Light reaction takes place on the membranes of the thylakoids. Chloroplasts, like the mitochondria use the potential energy of the H+ ions or the hydrogen ion gradient to generate energy in the form of ATP. The dark reactions also known as the Calvin cycle take place in the stroma of chloroplast. Production of NADPH2 molecules and oxygen as a result of photolysis of water. BY the utilization of assimilatory powers the 6-carbon atom is broken into two molecules of phosphoglyceric acid. Endoplasmic Reticulum Endoplasmic reticulum is a continuous membrane, which is present in both plant cells, animal cells and absent in prokaryotic cells. It is the membrane of network tubules and flattened sacs, which serves a variety of functions within the cell. The space, which is present in the endoplasmic reticulum, is called as the lumen.
- 12. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 11 2016-17 Endoplasmic Reticulum Structure Endoplasmic reticulum is an extensive membrane network of cisternae (sac-like structures), which are held together by the cytoskeleton. The phospholipid membrane encloses a space, the lumen from the cytosol, which is continuous with the perinuclear space. The surface of the rough endoplasmic reticulum is studded with the protein manufacturing ribosome, which gives it a rough appearance. Hence it is referred as a rough endoplasmic reticulum. The smooth endoplasmic reticulum consists of tubules, which are located near the cell periphery. This network increases the surface area for the storage of key enzymes and the products of these enzymes. Rough endoplasmic reticulum synthesizes proteins, while smooth endoplasmic reticulum synthesizes lipids and steroids. It also metabolizes carbohydrates and regulates calcium concentration, drug detoxification, and attachment of receptors on cell membrane proteins. The major functions of Endoplasmic reticulum are: 1. It is mainly responsible for the transportation of proteins and other carbohydrates to another organelle, which includes lysosomes, Golgi apparatus, plasma membrane, etc. 2. They play a vital role in the formation of the skeletal framework. 3. They provide the increased surface area for cellular reactions. 4. They help in the formation of nuclear membrane during cell division. 5. They play a vital role in the synthesis of proteins, lipids, glycogen and other steroids like cholesterol, progesterone, testosterone, etc. Ribosomes: Typically ribosomes are composed of two subunits: a large subunit and a small subunit. The subunits of the ribosome are synthesized by the nucleolus. The subunits of ribosomes join together when the ribosomes attaches to the messenger RNA during the process of protein synthesis. Ribosomes along with a transfer RNA molecule (tRNA), helps to translate the protein-coding genes in mRNA to proteins. Ribosome Structure Ribosomes in a cell are located in two regions of the cytoplasm. They are found scattered in the cytoplasm and some are attached to the endoplasmic reticulum.
- 13. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 12 2016-17 When the ribosomes are bound to the ER there are known as the rough endoplasmic reticulum. The bound and the free ribosomes are similar in structure and are invloved in protein synthesis. Ribosomes are tiny particles about 200 Ao Ribosomes are composed of both RNA and proteins. About 37 - 62% of RNA are made up of RNA and the rest is proteins. Ribosome is made up of two subunits. The subunits of ribosomes are named according to their ability of sedimentation on a special gel which the Sevdberg Unit. Prokarytotes have 70S ribosomes each subunit consisting of small subunit is of 30S and the large subunit is of 50S. Eukarytotes have 80S ribosomes each consisting of small (40S) and large (60S) subunit. The ribosomes found in the chloroplasts of mitochondria of eukaryotes consist of large and small subunits bound together with proteins into one 70S particle. The ribosomes share a core structure which is similar to all ribosomes despite differences in its size. The RNA is organized in various tertiary structures. The RNA in the larger ribosomes is into several continuous insertion as they form loops out of the core structure without disrupting or changing it. The catalytic activity of the ribosome is carried out by the RNA; the proteins reside on the surface and stabilize the structure. The differences between the ribosomes of bacterial and eukaryotic are used to create antibiotics that can destroy bacterial infection without harming human cells. Function The main functions of ribosome are: They assemble amino acids to form specific proteins, proteins are essential to carry out cellular activities. The process of production of proteins, the deoxyribonucleic acid produces mRNA by the process of DNA transcription. The genetic message from the mRNA is translated into proteins during DNA translation. The sequences of protein assembly during protein synthesis are specified in the mRNA. The mRNA is synthesized in the nucleus and is transported to the cytoplasm for further process of protein synthesis. In the cytoplasm, the two subunits of ribosomes are bound around the polymers of mRNA; proteins are then synthesized with the help of transfer RNA. The proteins that are synthesized by the ribosomes present in the cytoplasm are used in the cytoplasm itself. The proteins produced by the bound ribosomes are transported outside the cell.
- 14. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 13 2016-17 Golgi Apparatus Structure The Golgi apparatus is a major organelle in most of the eukarytoic cells. They are membrane bound organelles, which are sac-like. They are found in the cytoplasm of plant and animal cells. The Golgi complex is composed of stacks of membrane-bound structures, these structures are known as the cisternae. An individual stack of the cisternae is sometimes referred as dictyosome. In a typical animal cell, there are about 40 to 100 stacks. In a stack there are about four to eight cisternae. Each cisternae is a disc enclosed in a membrane, it possess special enzymes of the Golgi which help to modify and transport of the modified proteins to their destination. The flat sacs of the cisternae are stacked and are bent and semicircular in shape. Each group of stacks is membrane bound and its insides are separated from the cytoplasm of the cell. The interaction in the Golgi membrane in responsible for the unique shape of the apparatus. The Golgi complex is polar in nature. The membranes of one end of the stack are different in composition and thickness to the membranes at the other end. One end of the stack is known as the cis face, it is the 'receiving department" while the other end is the trans face and is the "shipping department". The cis face of the Golgi apparatus is closely associated with the endoplasmic reticulum. Function 1. The cell synthesize a huge amount of variety of macromolecules. The main function of the Golgi apparatus is to modify, sort and package the macromolecules that are synthesized by the cells for secretion purposes or for use within the cell. 2. It mainly modifies the proteins that are prepared by the rough endoplasmic reticulum. 3. They are also involved in the transport of lipid molecules around the cell. 4. They also create lysosomes. 5. The Golgi complex is thus referred as post office where the molecules are packaged, labelled and sent to different parts of the cell. 6. The enzymes in the cisternae have the ability to modify proteins by the addition of carbohydrates and phosphate by the process of glycosylation and phoshphorylation respectively. 7. In order to modify the proteins the golgi complex imports substances like nucleotides from the cytosol of the cell. The modifications brought about by the golgi body might form a signal sequence. This determines the final destination of the protein.
- 15. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 14 2016-17 8. The Golgi complex also plays an important role in the production of proteoglycans. The proteoglycans are molecules that are present in the extracellular matrix of the animal cells. 9. It is also a major site of synthesis of carbohydrates. These carbohydratres includes the synthesis of glycoasaminoglycans, Golgi attaches to these polysaccharides which then attaches to a protein produced in the endoplasmic reticulum to form proteoglycans. 10. The Golgi involves in the sulfation process of certain molecules. 11. The process of phosphorylation of molecules by the Golgi requires the import of ATP into the lumen of the Golgi. Lysosomes Lysosomes are single membrane bound structures. They are tiny sac like structures and are present all over the cytoplasm. The main function is digestion. They contain digestive enzymes. Lysosomes contain digestive enzymes that are acid hydrolases. They are responsible for the degrading of proteins and worn out membranes in the cell and also help degradation of materials that are ingested by the cell. Lysosomes that are present in the white blood cells are capable of digesting invading microorganisms like the bacteria and viruses. During the period of starvation the lysosomes digest proteins, fats and glycogen in the cytoplasm. They are capable of digesting the entire damaged cell containing them, hence, the lysosomes are known as "suicide bags" of the cell.
- 16. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 15 2016-17 Peroxisomes Peroxisomes are found in liver and kidney cells. Peroxisomes have enzymes that are responsible to get rid of the toxic peroxides from the cell. Peroxisomes (also called microbodies) are organelles found in virtually all eukaryotic cells. They are involved in catabolism of very long chain fatty acids, branched chain fatty acids, D- amino acids, and polyamines, reduction of reactive oxygen species – specifically hydrogen peroxide – and biosynthesis of plasmalogens, i.e. ether phospholipids critical for the normal function of mammalian brains and lungs. They also contain approximately 10% of the total activity of two enzymes in the pentose phosphate pathway, which is important for energy metabolism. Other known peroxisomal functions include the glyoxylate cycle in germinating seeds ("glyoxysomes"), photorespiration in leaves, glycolysis in trypanosomes ("glycosomes"), and methanol and/or amine oxidation and assimilation in some yeasts.
- 17. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 16 2016-17 Glyoxysomes: Glyoxysomes are specialized peroxisomes found in plants (particularly in the fat storage tissues of germinating seeds) and also in filamentous fungi. Seeds that contain fats and oils include corn, soybean, sunflower, peanut and pumpkin As in all peroxisomes, in glyoxysomes the fatty acids are oxidized to acetyl-CoA by peroxisomal β-oxidation enzymes. When the fatty acids are oxidized hydrogen peroxide (H2O2) is produced as oxygen (O2) is consumed. Thus the seeds need oxygen to germinate. Besides peroxisomal functions, glyoxysomes possess additionally the key enzymes of glyoxylate cycle (isocitrate lyase and malate synthase) which accomplish the glyoxylate cycle bypass. Thus, glyoxysomes (as all peroxisomes) contain enzymes that initiate the breakdown of fatty acids and additionally possess the enzymes to produce intermediate products for the synthesis of sugars by gluconeogenesis. The seedling uses these sugars synthesized from fats until it is mature enough to produce them by photosynthesis. Plant peroxisomes also participate in photorespiration and nitrogen metabolism in root nodules. They have single membrane. Their matrix is finely granular. Contains some enzymes like catalase fatty acid oxidase. Endosome: In cell biology, an endosome is a membrane-bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway originating from the trans Golgi membrane. Molecules or ligands internalized from the plasma membrane can follow this pathway all
- 18. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 17 2016-17 the way to lysosomes for degradation, or they can be recycled back to the plasma membrane. Molecules are also transported to endosomes from the trans-Golgi network and either continue to lysosomes or recycle back to the Golgi. Endosomes can be classified as early, sorting, or late depending on their stage post internalizationEndosomes represent a major sorting compartment of the endomembrane system in cells. In HeLa cells, endosomes are approximately 500 nm in diameter when fully mature. Function Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome For example, LDLis taken into the cell by binding to the LDL receptor at the cell surface. Upon reaching early endosomes, the LDL dissociates from the receptor, and the receptor can be recycled to the cell surface. The LDL remains in the endosome and is delivered to lysosomes for processing. LDL dissociates because of the slightly acidified environment of the early endosome, generated by a vacuolar membrane proton pump V-ATPase. On the other hand, EGF and the EGF receptor have a pH-resistant bond that persists until it is delivered to lysosomes for their degradation. The mannose 6-phosphate receptor carries ligands from the Golgi destined for the lysosome by a similar mechanism. Types Endosomes comprise three different compartments: early endosomes, late endosomes, and recycling endosomes. They are distinguished by the time it takes for endocytosed material to reach them, and by markers such as rabs They also have different morphology. Once endocytic vesicles have uncoated, they fuse with early endosomes. Early endosomes then mature into late endosomes before fusing with lysosomes. Early endosomes mature in several ways to form late endosomes. They become increasingly acidic mainly through the activity of the V-ATPase. Many molecules that are recycled are removed by concentration in the tubular regions of early endosomes. Loss of these tubules to recycling pathways means that late endosomes mostly lack tubules. They also increase in size due to the homotypic fusion of early endosomes into larger vesicles. Molecules are also sorted into smaller vesicles that bud from the perimeter membrane into the endosome lumen, forming lumenal vesicles; this leads to the multivesicular appearance of late endosomes and so they are also known as multivesicular bodies (MVBs). Removal of recycling molecules such as transferrin receptors and mannose 6- phosphate receptors continues during this period, probably via budding of vesicles out of endosomes.] Finally, the endosomes lose RAB5A and acquire RAB7A, making them competent for fusion with lysosomesFusion of late endosomes with lysosomes has been shown to result in the formation of a 'hybrid' compartment, with characteristics intermediate of the two source compartments. For example, lysosomes are more dense than late endosomes, and the hybrids have an intermediate density. Lysosomes reform by recondensation to their normal, higher density. However, before this happens, more late endosomes may fuse with the hybrid. Some material recycles to the plasma membrane directly from early endosomes but most traffics via recycling endosomes.
- 19. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 18 2016-17 Early endosomes consist of a dynamic tubular-vesicular network (vesicles up to 1 µm in diameter with connected tubules of approx. 50 nm diameter). Markers include RAB5Aand RAB4, Transferrin and its receptor and EEA1. Late endosomes, also known as MVBs, are mainly spherical, lack tubules, and contain many close-packed lumenal vesicles. Markers include RAB7, RAB9, and mannose 6-phosphate receptors.[11] Recycling endosomes are concentrated at the microtubule organizing center and consist of a mainly tubular network. Marker; RAB11. More subtypes exist in specialized cells such as polarized cells and macrophages. Phagosomes, macropinosomes and autophagosomes mature in a manner similar to endosomes, and may require fusion with normal endosomes for their maturation. Some intracellular pathogens subvert this process, for example, by preventing RAB7 acquisition. Late endosomes/MVBs are sometimes called endocytic carrier vesicles, but this term was used to describe vesicles that bud from early endosomes and fuse with late endosomes. However, several observations (described above) have now demonstrated that it is more likely that transport between these two compartments occurs by a maturation process, rather than vesicle transport. Another unique identifying feature that differs between the various classes of endosomes is the lipid composition in their membranes. Phosphotidyl inositol phosphates (PIPs), one of the most important lipid signaling molecules, is found to differ as the endosomes mature from early to late. PI(4,5)P2 is present on plasma membranes, PI(3)P on early endosomes, PI(3,5)P2 on late endosomes and PI(4)P on the trans Golgi network. These lipids on the surface of the endosomes help in the specific recruitment of proteins from the cytosol, thus providing them an identity. The inter- conversion of these lipids is a result of the concerted action of phosphoinositide kinases and phosphatases that are strategically localized Pathways Diagram of the pathways that intersect endosomes in the endocytic pathway of animal cells. Examples of molecules that follow some of the pathways are shown, including receptors for EGF, transferrin, and lysosomal hydrolases. Recycling endosomes, and compartments and pathways found in more specialized cells, are not shown. There are three main compartments that have pathways that connect with endosomes. More pathways exist in specialized cells, such as melanocytes and polarized cells. For example, in epithelial cells, a special process called transcytosis allows some materials to enter one side of a cell and exit from the opposite side. Also, in some circumstances, late endosomes/MVBs fuse with the plasma membrane instead of with lysosomes, releasing the lumenal vesicles, now called exosomes, into the extracellular medium. It should be noted that there is no consensus as to the exact nature of these pathways, and the sequential route taken by any given cargo in any given situation will tend to be a matter of debate. Golgi to/from endosomes
- 20. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 19 2016-17 Vesicles pass between the Golgi and endosomes in both directions. The GGAs and AP-1 clathrin-coated vesicle adaptors make vesicles at the Golgi that carry molecules to endosomes. In the opposite direction, retromer generates vesicles at early endosomes that carry molecules back to the Golgi. Some studies describe a retrograde traffic pathway from late endosomes to the Golgi that is mediated by Rab9 and TIP47, but other studies dispute these findings. Molecules that follow these pathways include the mannose-6-phosphate receptors that carry lysosomal hydrolases to the endocytic pathway. The hydrolases are released in the acidic environment of endosomes, and the receptor is retrieved to the Golgi by retromer and Rab9. Plasma membrane to/from early endosomes (via recycling endosomes) Molecules are delivered from the plasma membrane to early endosomes in endocytic vesicles. Molecules can be internalized via receptor-mediated endocytosis in clathrin-coated vesicles. Other types of vesicles also form at the plasma membrane for this pathway, including ones utilising caveolin. Vesicles also transport molecules directly back to the plasma membrane, but many molecules are transported in vesicles that first fuse with recycling endosomes.[18] Molecules following this recycling pathway are concentrated in the tubules of early endosomes. Molecules that follow these pathways include the receptors for LDL, the growth factor EGF, and the iron transport protein transferrin. Internalization of these receptors from the plasma membrane occurs by receptor- mediated endocytosis. LDL is released in endosomes because of the lower pH, and the receptor is recycled to the cell surface. Cholesterol is carried in the blood primarily by (LDL), and transport by the LDL receptor is the main mechanism by which cholesterol is taken up by cells. EGFRs are activated when EGF binds. The activated receptors stimulate their own internalization and degradation in lysosomes. EGF remains bound to the EGFR once it is endocytosed to endosomes. The
- 21. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 20 2016-17 activated EGFRs stimulate their own ubiquitination, and this directs them to lumenal vesicles (see below) and so they are not recycled to the plasma membrane. This removes the signaling portion of the protein from the cytosol and thus prevents continued stimulation of growth - in cells not stimulated with EGF, EGFRs have no EGF bound to them and therefore recycle if they reach endosomes. Transferrin also remains associated with its receptor, but, in the acidic endosome, iron is released from the transferrin, and then the iron-free transferrin (still bound to the transferrin receptor) returns from the early endosome to the cell surface, both directly and via recycling endosomes. Late endosomes to lysosomes Transport from late endosomes to lysosomes is, in essence, unidirectional, since a late endosome is "consumed" in the process of fusing with a lysosome. Hence, soluble molecules in the lumen of endosomes will tend to end up in lysosomes, unless they are retrieved in some way. Transmembrane proteins can be delivered to the perimeter membrane or the lumen of lysosomes. Transmembrane proteins destined for the lysosome lumen are sorted into the vesicles that bud from the perimeter membrane into endosomes, a process that begins in early endosomes. When the endosome has matured into a late endosome/MVB and fuses with a lysosome, the vesicles in the lumen are delivered to the lysosome lumen. Proteins are marked for this pathway by the addition of ubiquitin The endosomal sorting complexes required for transport (ESCRTs) recognise this ubiquitin and sort the protein into the forming lumenal vesicles. Molecules that follow these pathways include LDL and the lysosomal hydrolases delivered by mannose-6-phosphate receptors. These soluble molecules remain in endosomes and are therefore delivered to lysosomes. Also, the transmembrane EGFRs, bound to EGF, are tagged with ubiquitin and are therefore sorted into lumenal vesicles by the ESCRTs. Cytoskeleton: A cytoskeleton is present in all cells of all domains of life (archaea, bacteria, eukaryotes). It is a complex network of interlinking filaments and tubules that extend throughout the cytoplasm, from the nucleus to the plasma membrane. The cytoskeletal systems of different organisms are composed of similar proteins. In eukaryotes, the cytoskeletal matrix is a dynamic structure composed of three main proteins, which are capable of rapid growth or disassembly dependent on the cell's requirements at a certain period of time.[ The structure, function and dynamic behavior of the cytoskeleton can be very different, depending on organism and cell type. Even within one cell the cytoskeleton can change through association with other proteins and the previous history of the network. There is a multitude of functions that the cytoskeleton can perform. Primarily, it gives the cell shape and mechanical resistance to deformation, and through association with extracellular connective tissue and other cells it stabilizes entire tissues.
- 22. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 21 2016-17 The cytoskeleton can also actively contract, thereby deforming the cell and the cell's environment and allowing cells to migrate. Moreover, it is involved in many cell signaling pathways, in the uptake of extracellular material (endocytosis), segregates chromosomes during cellular division, is involved in cytokinesis (the division of a mother cell into two daughter cells), provides a scaffold to organize the contents of the cell in space and for intracellular transport (for example, the movement of vesicles and organelles within the cell); and can be a template for the construction of a cell wall. Furthermore, it forms specialized structures, such as flagella, cilia, lamellipodia and podosomes. A large-scale example of an action performed by the cytoskeleton is muscle contraction. During contraction of a muscle, within each muscle cell, myosin molecular motors collectively exert forces on parallel actin filaments. This action contracts the muscle cell, and through the synchronous process in many muscle cells, the entire muscle. Microfilaments: Microfilaments, also called actin filaments, are filamentous structures in the cytoplasm of eukaryotic cells and form part of the cytoskeleton. They are primarily composed of polymers of actin, but in cells are modified by and interact with numerous other proteins. Microfilament functions include cytokinesis, amoeboid movement and cell motility in general, changes in cell shape, endocytosis and exocytosis, cell contractility and mechanical stability. Microfilaments are flexible and relatively strong, resisting buckling by multi-piconewton compressive forces and filament fracture by nanonewton tensile forces. In inducing cell motility, one end of the actin filament elongates while the other end contracts, presumably by myosin II molecular motors.[1] Additionally, they function as part of actomyosin-driven contractile molecular motors, wherein the thin filaments serve as tensile platforms for myosin's ATP-dependent pulling action in muscle contraction and pseudopod advancement. Microfilaments have a tough, flexible framework which helps the cell in movement. Actin: Actin can hydrolyze its bound ATP to ADP + Pi, releasing Pi. The actin monomer can exchange bound ADP for ATP. The conformation of actin is different, depending on whether there is ATP or ADP in the nucleotide-binding site.
- 23. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 22 2016-17 G-actin (globular actin) with bound ATP can polymerize, to form F- actin (filamentous actin). F-actin may hydrolyze its bound ATP to ADP + Pi and release Pi. ADP release from the filament does not occur because the cleft opening is blocked. ADP/ATP exchange: G-actin can release ADP and bind ATP, which is usually present in the cytosol at higher concentration than ADP. Capping proteins bind at the ends of actin filaments. Different capping proteins may either stabilize an actin filament or promote disassembly. They may have a role in determining filament length. For example: Tropomodulins cap the minus end, preventing dissociation of actin monomers. CapZ capping protein binds to the plus end, inhibiting polymerization. If actin monomers continue to dissociate from the minus end, the actin filament will shrink. Cross-linking proteins organize actin filaments into bundles or networks. Actin-binding domains of several of the cross- linking proteins (e.g., filamin, a-actinin, spectrin, dystrophin and fimbrin) are homologous. Most cross-linking proteins are dimeric or have 2 actin-binding domains. Some actin-binding proteins such as a- actinin, villin and fimbrin bind actin filaments into parallel bundles. Depending on the length of a cross-linking protein, or the distance between actin-binding domains, actin filaments in parallel bundles may be held close together, or may be far enough apart to allow interaction with other proteins such as myosin. Filamins dimerize, through antiparallel association of their C-terminal domains, to form V-shaped cross-linking proteins
- 24. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 23 2016-17 that have a flexible shape due to hinge regions. Filamins organize actin filaments into loose networks that give some areas of the cytosol a gel-like consistency. Filamins may also have scaffolding rolesrelating to their ability to bind constituents of signal pathways such as plasma membrane receptors, calmodulin, caveolin, protein kinase C, transcription factors, etc. Spectrin is an actin-binding protein that forms an elongated tetrameric complex having an actin- binding domain at each end. With short actin filaments, spectrin forms a cytoskeletal network on the cytosolic surface of the plasma membrane of erythrocytes and some other cells. Cell structures that involve actin (selected examples): Filopodia (also called microspikes) are long, thin and transient processes that extend out from the cell surface. Bundles of parallel actin filaments, with their plus ends oriented toward the filopodial tip, are cross-linked within filopodia by a small actin-binding protein such as fascin. The closely spaced actin filaments provide stiffness. Microvilli are shorter and more numerous protrusions of the cell surface found in some cells. Tightly bundled actin filaments within these structures also have their plus ends oriented toward the tip. Small cross-linking proteins such as fimbrin and villin bind actin filaments together within microvilli. Lamellipodia are thin but broad projections at the edge of a mobile cell. Lamellipodia are dynamic structures, constantly changing shape. Stress fibers form when a cell makes stable connections to a substrate. o Bundles of actin filaments extend from the cell surface through the cytosol. The actin filaments, whose plus ends are oriented toward the cell surface on opposite sides of the cell, may overlap in more interior regions of a cell, in anti-parallel arrays o Myosin mediates sliding of anti-parallel actin filaments during contraction of stress fibers. o a-Actinin may cross-link actin filaments within stress fibers. Some cells have a cytoskeletal network just inside the plasma membrane that includes actin along with various other proteins such as spectrin. This cytoskeleton has a role in maintaining cell shape. An example is found in erythrocytes. Diagram & micrograph in A. p. 603. Actin filaments have an essential role in the contractile ring responsible for cytokinesis at the end of mitosis in animal cells. Diagram in A. p. 1054. Actin is found in the cell nucleus as well as in the cytoplasm. Recent data indicate involvement of actin in regulation of gene transcription.
- 25. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 24 2016-17 Microtubules: Microtubules are present in eukaryotic cells, including plant cells and animal cells. Microtubules are long thin structures that consist of the protein tubulin and typically have a diameter of about 25 nm. Characteristics of microtubules that are important for their functions include: Long rigid shape - which enables microtubules to support other structures within the cell Ability to generate movement - both within cells (see their role in moving centrioles during mitosis - below) and of the whole cells themselves (in the cases of microtubules that form structures such as cilia and flagella). Structures and Functions of Microtubules Microtubules are filamentous intracellular structures that are responsible for various kinds of movements in all eukaryotic cells. Microtubules are involved in nucleic and cell division, organization of intracellular structure, and intracellular transport, as well as ciliary and flagellar motility. "Building blocks" of microtubules - tubulins All eukaryotic cells produce the protein tubulin, in the usual way. The usual way, of course, is by transcription of genes coding for tubulin to produce messenger RNA, followed by the translation of mRNA by the ribosomes in order to produce protein. Cells maintain at least two types of tubulin, which we call alpha tubulin and beta tubulin. However, it is doubtful that the two types can found in cells as individual proteins. Alpha and beta tubulin spontaneously bind one another to form a functional subunit that we call a heterodimer. A heterodimer is a protein that consists of two different gene products. The term is entirely descriptive - the prefix hetero- means "different," the prefix di- means "two," and the suffix - mer refers to a unit, in this case a single polypeptide. Obviously, cells do not continue to make tubulin
- 26. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 25 2016-17 (or any other protein) until they run out of resources. Some process must regulate the synthesis of tubulin. A common regulatory mechanism is feedback inhibition. The figure illustrates the inhibition of tubulin synthesis by the presence of heterodimers in the system. Exactly how that inhibition takes place is irrelevant to this discussion. Assembly of microtubules When intracellular conditions favor assembly, tubulin heterodimers assemble into linear protofilaments. Protofilaments in turn assemble into microtubules. All such assembly is subject to regulation by the cell. Microtubules form a framework for structures such as the spindle apparatus that appears during cell division, or the whiplike organelles known as cilia and flagella. Cilia and flagella are the most well-studied models for microtubule structure and assembly, and are often used by textbooks to introduce microtubules. Dynamic instability of microtubules Under steady state conditions a microtubule may appear to be completely stable, however there is action taking place constantly. Populations of microtubules usually consist of some that are shrinking and some that are growing. A single microtubule can oscillate between growth and shortening
- 27. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 26 2016-17 phases. During growth, heterodimers are added on to the end of a microtubule, and during shrinkage they come off as intact subunits. The same heterodimer can come off and go back on. Since even apparently stable microtubular structures have an intrinsic instability, they are considered to be in a dynamic equilibrium, or steady state. Look here to learn about the difference between a steady state and a true equilibrium. Microtubules consist of longitudinally arranged assemblies of filaments called protofilaments. These protofilaments group together to form long cylinders or tubes called microtubules such that a cross- sectional view of a microtubule would look like a circle formed by the cross-sections (diameters) of approximately thirteen (13) protofilaments. The protofilaments themselves have two forms - they are both molecules of the protein tubulin but they differ in the sequence in which the amino acids forming the protein are arranged. The two types of protofilaments are: alpha tubulin (sometimes written α-tubulin) and beta tubulin (sometimes written β-tubulin) 1. Microtubules are an important part of the cytoskeleton of cells Electron microscopy has revealed that cells contain a network of interconnected fibres and filaments that can be observed as thread-like structures when viewed using an electron microscope or fluorescence microscope (tagged with antibodies and fluorescent dyes). This network is called the cytoskeleton. It extends throughout the cell and connects with the cell membrane and organelles.
- 28. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 27 2016-17 The main functions of the cytoskeleton of a cell are: 1. Helps maintain and control the shape of the cell. 2. Aids communication between organelles in the cell. 3. Aids cyclosis, i.e. movement of the cytoplasmwithin cells - sometimes described as "cyclical streaming" of the cytoplasm (in plant cells). 4. Aids movement of certain structures within the cell, such as chromosomes, granules and membranes (e.g. movement of membranes to form protrusions such as the microvilli of some animal cells). 2. Role of microtubules during the prophase stage of mitosis The two diagrams below illustrate the prophase stage of mitosis in which the following occurs: 1. Early in the prophase stage the chromatin fibres shorten into chromosomes that are visible under a light microscope. (Each prophase chromosome consists of a pair of identical double- stranded chromatids.) 2. Later in prophase, the nucleolus disappears, the nuclear envelope breaks down, and the two centrosomes begin to form the miotic spindle (which is an assembly of microtubules). 3. As the microtubules extend in length between the centrosomes, the centrosomes are pushed to opposite "poles" (extremes) of the cell. 4. Eventually, the spindle extends between two opposite poles of the cell, as shown below. Early Prophase Late Prophase
- 29. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 28 2016-17 The mitotic spindle is formed during prophase and remains within the dividing cell, an important part of its structure, through metaphase and anaphase until it finally breaks-up when the two new cells separate during telophase. Intermediate filaments Intermediate filaments are a primary component of the cytoskeleton, although they are not found in all eukaryotes, and are absent in fungi and plants . These filaments, which extend throughout the cytoplasm and inner nuclear membrane are composed from a large family of proteins that can be broadly grouped into five classes. IF assembly begins with the folding of IF proteins into a conserved alpha-helical rod shape, followed by a series of polymerization and annealing events that lead to the formation of filaments roughly 8 to 12 nm in diameter. Different IF combinations are found in different cell types, however not all IF classes will interact with each other. In contrast to other cytoskeletal components (e.g. actin filaments, microtubules), intermediate filaments lack polarity, are more stable and their constituent subunits do not bind nucleotides (such as ATP). Function of intermediate filaments The tight association between protofilaments provide intermediate filaments with a high tensile strength. This makes them the most stable component of the cytoskeleton. Intermediate filaments are therefore found in particularly durable structures such as hair, scales and fingernails. The primary function of intermediate filaments is to create cell cohesion and prevent the acute fracture of epithelial cell sheets under tension. This is made possible by extensive interactions between the constituent protofilaments of an intermediate filament, which enhance its resistance to compression, twisting, stretching and bending forces. These properties also allow intermediate filaments to help stabilize the extended axons of nerve cells, as well as line the inner face of the nuclear envelope where they help harness and protect the cell’s DNA.
- 30. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 29 2016-17 Types There are about 70 different genes coding for various intermediate filament proteins. However, different kinds of IFs share basic characteristics: In general, they are all polymers that measure between 9-11 nm in diameter when fully assembled. IF are subcategorized into six types based on similarities in amino acid sequence and protein structure. Types I and II – acidic and basic keratins These proteins are the most diverse among IFs and constitute type I (acidic) and type II (basic) IF proteins. The many isoforms are divided in two groups: epithelial keratins (about 20) in epithelial cells (image to right) trichocytic keratins (about 13) (hair keratins), which make up hair, nails, horns and reptilian scales. Regardless of the group, keratins are either acidic or basic. Acidic and basic keratins bind each other to form acidic-basic heterodimers and these heterodimers then associate to make a keratin filament. Type III There are four proteins classed as type III IF proteins, which may form homo- or heteropolymeric proteins. Desmin IFs are structural components of the sarcomeres in muscle cells. GFAP (glial fibrillary acidic protein) is found in astrocytes and other glia. Peripherin found in peripheral neurons. Vimentin, the most widely distributed of all IF proteins, can be found in fibroblasts, leukocytes, and blood vessel endothelial cells. They support the cellular membranes, keep some organelles in a fixed place within the cytoplasm, and transmit membrane receptor signals to the nucleus. Type IV α-Internexin Neurofilaments - the type IV family of intermediate filaments that is found in high concentrations along the axons of vertebrate neurons. Synemin Syncoilin Type V - nuclear lamins Lamins
- 31. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 30 2016-17 Lamins are fibrous proteins having structural function in the cell nucleus. Type VI Nestin Marker enzymes: Golgi markers The main function for Golgi apparatus is the proper folding of macromolecules and their secretion to the extracellular environment (exocytosis via the trans-Golgi network) or in their transport, together with other proteins and lipids, in the intracellular environment (Golgi stack). Golgis can also
- 32. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 31 2016-17 synthesize proteoglycans and carbohydrates. They are usually disassembled during mitosis and reassembled again after mitosis in each daughter cell. RCAS1 or EBAG9 The receptor binding cancer antigen expressed on SiSo cells is a type III transmembrane Golgi protein and more specifically localized at ER-Golgi intermediate compartment and the cis-Golgi Syntaxin 6 is mainly found in Golgi and is involved in trafficking of intracellular vesicles. Formimidoyltransferase-cyclodeaminase (FTCD) is a 58 kDa enzyme with transferase and deaminase activity. It is localized on Golgi and facilitates bundling of vimentin starting from Golgi, but recently has been also found in the centrosome. Golgin subfamily A member 2 (or GM130) is a Golgi auto-antigen which probably has a function in ER-Golgi transport. Alpha-mannosidase II is localized on the membrane of the Golgi apparatus and is involved in protein glycosylation, as it regulates steps of N-glycan synthesis. Beta1,4-galactosyltransferase 6 (b4Gal-T6) is involved in the biosynthesis of glucosphingolipids and is another Golgi internal membrane marker. TGN38 regulates membrane traffic from the trans-Golgi network (the secretory mechanism) to the plasma membrane. Upon Brefeldin A treatment, the Golgi stack is de-organized and the trans-Golgi network collapses upon the centrosome. Thus, TGN38 staining distinguishes the TGN from the Golgi stack. Mitochondrial markers The mitochondrion is an organelle of 0.5-1.0 μm in diameter. They are considered as the cellular power plants because they synthesize energy in the form of Adenosine Triphosphate (ATP) but they also have other functions. The mitochondrion is composed of the inner and outer membranes, the inter-membrane space, the cristae and the matrix while they contain their own DNA separated from the nuclear. In humans, more than 600 distinct proteins have been found and some of them are used as markers. Carbamoyl phosphate synthase I (CPS1) is the 163 kDa mitochondrial isozyme of this enzyme, which is involved in urea cycle and removes excess of ammonia from the cell. CPS1 is a marker of liver and kidney mitochondria. Prohibitin is a 30 kDa protein of the inner mitochondrial membrane and probably regulates mitochondrial respiration. It is involved in several activities including apoptosis, cell cycle regulation and senescence. Prohibitin is more abundant during G1 phase of the cell cycle and upon treatment with thiampenicol, a mitochondrial protein synthesis inhibitor. Cytochrome C oxidase (COX)
- 33. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 32 2016-17 is protein complex of the inner mitochondrial membrane [14]. It is involved in the translocation of protons and catalysis of oxygen to water required for ATP synthesis. Most of the COX subunits can be good mitochondrial markers. Apoptosis-inducing factor (AIF, PDCD8) is a 67 kDa protein of the inter-membrane space and is ubiquitously expressed. While in mitochondria, AIF functions as an oxidoreductase and has anti-apoptotic activity. However, during apoptotic signals, AIF is released to the cytoplasm and translocates to the nucleus to induce nuclear apoptosis. Hexokinase is a 100 kDa kinase of the outer mitochondrial membrane catalyzing the first step of glycolysis. Hexokinase phosphorylates hexoses (a six-carbon sugar) to form hexose phosphates (e.g. glucose to glucose-6-phosphate). VDAC1 (outer mitochondrial membrane protein porin 1) is the outer mitochondrial membrane receptor for hexokinase and BCL2L1. Nuclear markers There are several proteins used to distinguish the distinct nuclear structures from each other: Chromatin: The DNA molecule is condensed as it is wrapped around the four core histones (H2A, H2B, H3, H4 - which form an octamer) and form the nucleosome. Histone H1 is a protein linker which binds to distant chromatin areas and compacts it further. The DNA with its histones and other proteins that associated with it (to regulate transcription, replication, DNA repair etc) is called chromatin. Therefore, any dye that binds DNA can be used as a chromatin marker. For example, 4',6- diamidino-2-phenylindole (DAPI) and the dyes Hoechst 33258 and Hoechst 33342 are the most common. Bromodeoxyuridine (BrdU) is a synthetic nucleoside used to detect proliferating cells. 5- ethynyl-2'-deoxyuridine (EdU) staining is equally effective. All core histones are required for nucleosome formation. Therefore, anti-histone antibodies would mark mainly chromatin. Histone modification specific antibodies can distinguish between euchromatin (e.g. H3K4me3 or H3K36me3) and heterochromatin (e.g. H4K20me3). Phosphohistone H3 can also be used to indicate the mitotic state of cells. Telomeric repeat-binding factor 2-interacting protein 1 (TERF-1, RAP1) is localized at telomeres and regulates telomere length. Nuclear envelope: The nuclear envelope is the lipid bilayer membranous structure surrounding the nucleus and separates it from the cytoplasm. The inner membrane is comprised of a network of intermediate filaments, the lamina, while the outer membrane is physically linked to the endoplasmic reticulum, thus sharing some common proteins. Small holes on the nuclear envelope constitute the nuclear pore complexes of about 100 nm diameter and they connect the inner with the outer nuclear membrane as well as import or export proteins to and from the nucleus.
- 34. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 33 2016-17 Lamin A (74 kDa) and Lamin C as well as Lamin B (68 kDa) can be detected by antibodies to show the nuclear envelope, which is disassembled during mitosis. During apoptosis, lamin A and C will be cleaved into two fragments of 40-50 kDa and of 28 kDa. Nucleoporin 98 (NUP98) belongs to the nuclear pore complex. Nucleolus: The nucleolus is a non-membranous structure inside the nucleus of the cell which transcribes and assembles ribosomal RNAs. The protein components of the nucleoli can be used as markers, such as the RNA polymerase PAF49, the nucleolar protein 1 (Nop1p/Fibrillarin), Nop2p, Nop5p and Nsr1p. Endosomal markers Endosomes are cytoplasmic compartments which encircle molecules and transfer them from the membrane to other parts in the cell. Usually, endocytosed complexes (e.g. receptor-ligand) are separated in early endosomes and each component can be transferred to its new destination (e.g. lysosomes, Golgi etc.) via the late endosomes. Rab5, Rab7, Rab9 and Rab11 Rab5 is a small GTPase (24 kDa) of the Ras family which shuttles from the plasma membrane to early endosomes and regulates vesicular trafficking and fusion of plasma membranes with early endosomes, via its interaction with other proteins. Similarly, Rab7, Rab9 and Rab11 are equally good endosomal protein markers. EEA1 One of Rab5 interacting proteins is the early endosome antigen 1 (EEA1). It is a 162 kDa protein and participates in endosomal trafficking. Clathrin and Adaptor protein-2 (AP-2) Other structures for endocytosis and transfer of molecules are the clathrin-coated pits (or vesicles). These vesicles are consisted of a proteinaceous coat which packs membrane receptors and other molecules. Clathrin and Adaptor protein-2 (AP-2) are excellent markers of clathrin-coated vesicles. Exosomal markers Sometimes endosomes fuse with the plasma membrane and are secreted into the extracellular environment and they constitute a secretion mechanism. Exosomes are important as they contain cytoplasmic and membrane proteins as well as lipids and RNA molecules that might be potential biomarkers of particular diseases. HSPA8, and the tetraspanin proteins CD81 and CD9 are good markers of exosomes. Endoplasmic reticulum markers The endoplasmic reticulum (ER) is a cytoplasmic structure containing many chaperones that help polypeptides to fold properly and to assemble protein complexes. Most ER proteins contain the KDEL motif (Lysine-Aspartate-Glutamine-Leucine) and are retained through interaction with an internal ER KDEL receptor. Therefore, an anti-KDEL antibody recognizing the motif is used as ER positive marker. Calnexin is a 90 kDa integral ER membrane protein which binds unfolded proteins and retains them at ER. Calreticulin
- 35. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 34 2016-17 is 48 kDa chaperone of the ER with a KDEL motif at the C-term and binds monoglucosylated proteins synthesized in the ER. GRP 78 The 78 kDa glucose regulated protein (GRP 78) contains a KDEL C-term motif and facilitates the assembly of protein complexes in the ER. It is essential for cell viability. Protein disulfide-isomerase (PDI) PDI habitat is the ER due to its KDEL domain. PDI has several functions, including the formation of disulphide (S-S) bonds on unfolded proteins. Cytoskeletal markers Microtubules: Microtubules are elongated filaments consisting of tubulins. Alpha-tubulin and β-tubulin are globular proteins of 55 kDa which form heterodimers and they polymerize to form the cylindrical microtubule. They are involved in numerous cellular functions and especially cell structure maintenance, intracellular transport, or the formation of mitotic spindles that separate the sister chromatids during cell division. Tubulin polymerization starts at the centrosome, which constitutes the microtubule organizing centre (MTOC) in interphase and the spindle poles during mitosis, where distinct protein complexes constitute the scaffold for tubulin polymerization initiation. These complexes contain γ-tubulin. The centrosome has only one copy per cell, which will duplicate during mitosis. Antibodies targeting α- or β-tubulin are good markers for microtubules. Anti-γ-tubulin antibodies show the centrosome. Other centrosomal markers are the Pericentrin and Ninein. Pericentrin is a 220 kDa protein involved in the initial formation (nucleation) of the microtubule. Ninein is a centrosomal protein involved in microtubule nucleation and capping of the minus- and plus-ends. Actin filaments: Similarly to microtubules, actin filaments are double helical thin cylindrical tubes made of α- or β- actin. Their dynamic polymerization and depolymerization cycles regulate cell movement, cell polarization and scaffolding of the cell. In addition, several actin-binding proteins control actin polymerization. Anti-actin antibodies that bind to actin monomers or the fluorescently labeled toxin phalloidin that binds to filamentous actin. Autophagosomes and lysosomes Autophagosomes are intracellular organelles formed by elongation of small membrane structures, the autophagosome precursors, into a double membrane which surrounds damaged organelles. The autophagosome would then fuse with a lysosome, which contains hydrolases and other degradation enzymes, and is set for degradation. Apg12 and Apg5 are covalently associated with each other and function as a unit. Therefore, the Apg12-Apg5 conjugate is localized at the autophagosome membrane during elongation. Therefore it is a good marker for the initiation of autophagy. Several other Apg isoforms regulate autophagosome formation and its fusion to the lysosome and they can be used as autophagosomal markers.
- 36. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 35 2016-17 The microtubule-associated protein 1 light chain 3 (LC3) is also localized at the membrane but when fully formed it is found at the isolation membranes as well. LC3 can be found on lysosomes but with less abundance. Caution has been raised as whether Western blot of LC3 and its associated protein sequestosome 1 (SQSTM1, also known as p62) can truly reflect the status of autophagosomes, especially in the case of p62. Lysosome Associated Membrane Protein 1 and 2 (LAMP1; LAMP2) are components of the lysosomal membrane and therefore constitute excellent lysosomal markers. They have been recently used to confirm that transmembrane 7 superfamily member 1 (TM7SF1) is a protein of the integral membrane of the lysosome. Beta-galactosidase is one of the glycosidases that can be used as lysosomal markers. Cation-dependent mannose-6-phosphate receptor (M6PR) is involved in the transport of lysosomal enzymes from the Golgi and cell surface to the lysosomes. Melanosomal markers Melanosomes are organelles of melanocytes, skin and retina epithelial cells. Melanosomes contain the pigment melanin which protects cells from harmful ultraviolet (UV) radiation. Markers for melanosomes can be tyrosinase, Tyrp1, Dct, OA1, gp100, and MART1 [20]. Peroxisomal markers Peroxisomes are structures which are housing oxidative reactions, such as fatty acid β-oxidation, and protect from peroxides. Fox2p and Fox5p are peroxisomal membrane receptors. Catalase is a peroxisomal protein which protects cells from the toxic effects of hydrogen peroxide. Acyl-coenzyme A thioesterase 8 belongs to a group of enzymes that catalyze the hydrolysis of acyl- CoAs to the free fatty acid and coenzyme A (CoASH), providing the potential to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. Peroxins (a class of 24 genes) are integral parts of peroxisome development. Ribosomal markers Ribosomes are molecular complexes of proteins and RNA molecules (ribonucleoprotein) in which proteins are synthesized. They are comprised of a small 40S subunit and a large 60S subunit. Several ribosome-specific proteins can be used as markers. Antibodies against ribosomal proteins L7a, L26 (component of the 60 S subunit), S3, S6, S10, S11 (40S subunit) are characteristic examples. Proteasomal markers Proteasomes are multi-protein complexes and their function is to degrade proteins by proteolysis. Each proteasome consists of four stacked rings forming a central pore, the core. A seven-protein complex (β subunits) of proteolytic enzymes forms the two rings in the interior while seven α subunits form the entrance through which proteins enter and reach the core. The proteasome subunits 20S proteasome, 26S proteasome, α7 and Rpn2, Pre6, Cim5 and Scl1 are commonly used as proteasomal markers. Cell cycle markers Mitosis markers
- 37. Ultra structure of cell, By, sardar Hussain, Asst. Prof. GSC, CTA. 36 2016-17 Histone H3 is phosphorylated at Serine 10 (H3S10ph) and is essential for the onset of mitosis. Cytokinesis markers Aurora B kinase