lecture 3 b Cytoplasm and inclusion.pdf
- 1. Cytoplasm • The cytoplasm comprises cytosol – the gel-like substance enclosed within the cell membrane – and the organelles – the cell's internal sub-structures and the cytoskeleton • Cytosol • The cytosol is the portion of the cytoplasm not contained within membrane- bound organelles • Cytosol makes up about 70% of the cell volume and is composed of water, salts and organic molecules • The cytosol also contains the protein filaments that make up the cytoskeleton, as well as soluble proteins and small structures such as ribosomes, proteasomes
- 2. Cytoskeleton • The cytoskeleton is composed of three well-defined filamentous structures— microtubules, microfilaments, and intermediate filaments • Together these form an elaborate interactive network • Each of the three types of cytoskeletal filaments is a polymer of protein subunits held together by weak, non-covalent bonds • Each cytoskeletal element has distinct properties • Microtubules are long, hollow, unbranched tubes composed of subunits of the protein tubulin • Microfilaments are solid, thinner structures, often organized into a branching network and composed of the protein actin • Intermediate filaments are tough, rope-like fibers composed of a variety of related proteins
- 4. Functions of the cytoskeleton 1. Provide structural support that can determine the shape of the cell and resist forces that tend to deform it 2. Responsible for positioning the various organelles within the interior of the cell. This function is particularly evident in polarized epithelial cells, in which certain organelles are arranged in a defined order from the apical to the basal end of the cell 3. Direct the movement of materials and organelles within cells • E.g. the delivery of mRNA molecules to specific parts of a cell, the movement of membranous carriers from the endoplasmic reticulum to the Golgi complex, and the transport of vesicles containing neurotransmitters down the length of a nerve cell • Microtubules are the tracks over which the peroxisomes are transported in mammalian cells 4. The force-generating apparatus that moves cells from one place to another Single-celled organisms move either by “crawling” over the surface of a solid substratum or by propelling themselves through their aqueous environment with the aid of specialized, microtubule-containing locomotor organelles (cilia and flagella) that protrude from the cell’s surface
- 5. Functions of Cytoskeleton • Multicellular animals have a variety of cells that are capable of independent locomotion, including sperm, white blood cells, and fibroblasts (Figure 9.3). • The tip of a growing axon is also highly motile and its movement resembles a crawling cell 5. An essential component of the cell’s division machinery • Cytoskeletal elements make up the apparatus responsible for separating the chromosomes during mitosis and meiosis and for splitting the parent cell into two daughter cells during cytokinesis
- 7. MICROTUBULES • Structure and Composition • Microtubules are hollow, relatively rigid, tubular structures, and they occur in nearly every eukaryotic cell • Microtubules are components of many structures, including the mitotic spindle of dividing cells and the core of cilia and flagella • Microtubules have an outer diameter of 25 nm and a wall thickness of approximately 4 nm, • They may extend across the length or breadth of a cell • The wall of a microtubule is composed of globular proteins arranged in longitudinal rows, known proto-filaments, that are aligned parallel to the long axis of the tubule • Cross-section view reveal that microtubules consist of 13 proto-filaments aligned side by side in a circular pattern within the wall • Non-covalent interactions between adjacent proto-filaments play an important role in maintaining microtubule structure • Each proto-filament is assembled from dimeric building blocks consisting of one a-tubulin and one b-tubulin subunit
- 8. Microtubules: Structure and composition • The two types of globular tubulin subunits have a similar three-dimensional structure and fit tightly together • The tubulin dimers are organized in a linear array along the length of each proto-filament • Because each assembly unit contains two non-identical components (a heterodimer), the protofilament is asymmetric, with an a-tubulin at one end and a b-tubulin at the other end. • All of the protofilaments of a microtubule have the same polarity • Consequently, the entire polymer has polarity • One end of a microtubule is known as the plus end and is terminated by a row of b-tubulin subunits • The opposite end is the minus end and is terminated by a row of a- tubulin subunits • The structural polarity of microtubules is an important factor in the growth of these structures and their ability to participate in directed mechanical activities
- 9. Microtubule proteins • Microtubules from living tissue typically contain additional proteins, called microtubule-associated proteins (or MAPs) • MAPs comprise a heterogeneous collection of proteins • The first MAPs to be identified are referred to as “classical MAPs” and typically have one domain that attaches to the side of a microtubule and another domain that projects outward as a filament from the microtubule’s surface • Some MAPs can be seen in electron micrographs as cross-bridges connecting microtubules to each other, thus maintaining their parallel alignment • MAPs generally increase the stability of microtubules and promote their assembly • The microtubule-binding activity of the various MAPs is controlled primarily by the addition and removal of phosphate groups from particular amino acid residues • An abnormally high level of phosphorylation of one particular MAP, called tau, has been implicated in the development of several fatal neurodegenerative disorders, including Alzheimer’s disease
- 10. Microtubule proteins • The brain cells of people with these diseases contain strange, tangled filaments (called neurofibrillary tangles) consisting of tau molecules that are excessively phosphorylated and unable to bind to microtubules • The neurofibrillary filaments are thought to contribute to the death of nerve cells • Persons with one of these diseases, an inherited dementia called FTDP-17, carry mutations in the tau gene, indicating that alteration of the tau protein is the primary cause of this disorder
- 11. Functions of microtubules: Support • Microtubules are stiff enough to resist forces that might compress or bend the fiber • This property enables them to provide mechanical support • The distribution of cytoplasmic microtubules in a cell helps determine the shape of that cell • In cultured animal cells, the microtubules extend in a radial array outward from the area around the nucleus, giving these cells their round, flattened shape • In contrast, the microtubules of columnar epithelial cells are typically oriented with their long axis parallel to the long axis of the cell (Figure 9.1a) • This configuration suggests that microtubules help support the cell’s elongated shape • The role of microtubules as skeletal elements is particularly evident in certain highly elongated cellular processes, such as cilia and flagella and the axons of nerve cells • Microtubules also play a key role in maintaining the internal organization of cells
- 12. Functions of microtubules: Organization • Treatment of cells with microtubule-disrupting drugs can seriously affect the location of membranous organelles, including the ER and Golgi complex • The Golgi complex is typically located near the center of an animal cell, just outside the nucleus • Treatment of cells with nocodazole or colchicine, which promote microtubule • disassembly, can disperse the Golgi elements into the peripheral regions of the cell • When the drug is removed and microtubules reassemble, the Golgi membranes return to their normal position in the cell interior • Functions of microtubules: Intracellular motility • The transport of materials from one membrane compartment to another depends on the presence of microtubules because specific disruption of these cytoskeletal elements brings the movements to a halt • For e.g.intracellular motility by focusing on nerve cells whose intracellular movements rely on a highly organized arrangement of microtubules and other cytoskeletal filaments.
- 13. Functions of microtubules: Intracellular motility • Axonal Transport The axon of an individual motor neuron may stretch from the spinal cord to the tip of a finger or toe • The manufacturing center of this neuron is its cell body, a rounded portion of the cell that resides within the spinal cord • When labeled amino acids are injected into the cell body, they are incorporated into labeled proteins that move into the axon and gradually travel down its length • Many different materials, including neurotransmitter molecules, are compartmentalized within membranous vesicles in the ER and Golgi complex of the cell body and then transported down the length of the axon • Non-membrane-bound cargo, such as RNAs, ribosomes, and even cytoskeletal elements are also transported along this vast stretch of extended cytoplasm • Different materials move at different rates, with the fastest axonal transport occurring at velocities of 5 m per second • At this rate, a synaptic vesicle pulled by nanosized motor proteins can travel 0.4 meter in a single day, or about halfway from your spinal cord to the tip of your finger • Structures and materials traveling from the cell body toward the terminals of a neuron are said to move in an anterograde direction • Other structures, including endocytic vesicles that form at the axon terminals and carry regulatory factors from target cells, move in the opposite, or retrograde, direction—from the synapse toward the cell body • Defects in both anterograde and retrograde transport have been linked to several neurological diseases
- 14. INTERMEDIATE FILAMENTS • The second of the three major cytoskeletal elements to be discussed was seen in the electron microscope as solid, unbranched filaments with a diameter of 10–12 nm. • They were named intermediate filaments (or IFs). To date, intermediate filaments have only been identified in animal cells. • Intermediate filaments are strong, flexible ropelike fibers that provide mechanical strength to cells that are subjected to physical stress, including neurons, muscle cells, and the epithelial cells that line the body’s cavities. • Unlike microfilaments and microtubules, IFs are a chemically heterogeneous group of structures that, in humans, are encoded by approximately 70 different genes. • The polypeptide subunits of Ifs can be divided into five major classes based on the type of cell in which they are found (Table 9.2) as well as biochemical, genetic, and immunologic criteria. • We will restrict the present discussion to classes I-IV, which are found in the construction of cytoplasmic filaments
- 15. Intermediate filaments • IFs radiate through the cytoplasm of a wide variety of animal cells and are often interconnected to other cytoskeletal filaments by thin, wispy cross- bridges (Figure 9.40). • In many cells, these cross-bridges consist of an elongated dimeric protein called plectin that can exist in numerous isoforms. • Each plectin molecule has a binding site for an intermediate filament at one end and, depending on the isoform, a binding site for another intermediate filament, microfilament, or microtubule at the other end. • Although IF polypeptides have diverse amino acid sequences, all share a similar structural organization that allows them to form similar-looking filaments. Most notably, the polypeptides of IFs all contain a central, rod- shaped, -helical domain of similar length and homologous amino acid sequence. • This long fibrous domain makes the subunits of intermediate filaments very different from the globular tubulin and actin subunits of microtubules and microfilaments. • The central fibrous domain is flanked on each side by globular
- 17. Properties and Distribution of the Major Mammalian Intermediate Filament Proteins
- 18. Types and Functions of Intermediate Filaments • Keratin filaments constitute the primary structural proteins of epithelial cells (including epidermal cells, liver hepatocytes, and pancreatic acinar cells) • Figure 9.43a shows a schematic view of the spatial arrangement of the keratin filaments of a generalized epithelial cell, and Figure 9.43b shows the actual • arrangement within a pair of cultured epidermal cells. • Keratin-containing IFs radiate through the cytoplasm, tethered to the nuclear envelope in the center of the cell and anchored at the outer edge of the cell by connections to the cytoplasmic plaques of desmosomes and hemidesmosomes (pages 251 and 244). • Figure 9.43a also depicts the interconnections between IFs and the cell’s microtubules and microfilaments, which transforms these otherwise separate elements into an integrated cytoskeleton. • Because of these various physical connections, the IF network is able to serve as a scaffold for organizing and maintaining cellular architecture and for absorbing mechanical stresses applied by the extracellular environment.
- 19. IFs • The cytoplasm of neurons contains loosely packed bundles of intermediate filaments whose long axes are oriented parallel to that of the nerve cell axon (see Figure 9.13b). • These IFs, or neurofilaments, as they are called, are composed of three distinct proteins: NF-L, NF-H, and NF-M, all of the type IV group of Table 9.2. Unlike the polypeptides of other • IFs, NF-H and NF-M have sidearms that project outward from the neurofilament. These sidearms are thought to maintain the proper spacing between the parallel neurofilaments of the axon (see Figure 9.13b). • In the early stages of differentiation when the axon is growing toward a target cell, it contains very few neurofilaments but large numbers of supporting microtubules. Once the nerve cell has become fully extended, it becomes filled with neurofilaments that provide support as the axon increases dramatically in diameter. • Aggregation of NFs is seen in several human neurodegenerative disorders, including ALS and Parkinson’s disease. These NF aggregates may block axonal transport, leading to the death of affected neurons.
- 20. Ifs functions • Efforts to probe IF function have relied largely on genetically engineered mice that fail to produce a particular IF polypeptide (a gene knockout) or produce an altered IF polypeptide. • These studies have revealed the importance of intermediate filaments in particular cell types. For example, mice carrying deletions in the gene encoding K14, a type I keratin polypeptide normally synthesized by cells of the basal epidermal layer, have serious health problems. • These mice are so sensitive to mechanical pressure that even mild trauma, such as that occurring during passage through the birth canal or during nursing by the newborn, can cause severe blistering of the skin or tongue. This phenotype bears strong resemblance to a rare skin-blistering disease in humans, called epidermolysis bullosa simplex (EBS).3 Subsequent analysis of • EBS patients has shown that they carry mutations in the gene that encodes the homologous K14 polypeptide (or the K5 polypeptide, which forms dimers with K14). • These studies confirm the role of IFs in imparting mechanical strength to cells situated in epithelial layers.
- 21. Ifs functions • Similarly, knockout mice that fail to produce the desmin polypeptide exhibit serious cardiac and skeletal muscle abnormalities. • Desmin plays a key structural role in maintaining the alignment of the myofibrils of a muscle cell, and the absence of these IFs makes the cells extremely fragile. • An inherited human disease, named desminrelated myopathy, is caused by mutations in the gene that encodes desmin. • Persons with this disorder suffer from skeletal muscle weakness, cardiac arrhythmias, and eventual congestive heart failure. • Not all IF polypeptides have such essential functions. For example, mice that lack the vimentin gene, which is expressed in fibroblasts, macrophages, and white blood cells, show relatively minor abnormalities, even though the affected cells lack cytoplasmic IFs. • It is evident from these studies that IFs have tissue-specific functions, which are more important in some cells than in others.