Microfilaments and intermediate filaments
- 1. Department of Natural Sciences University of St. La Salle Bacolod City
- 2. CYTOSKELETON The cytoskeleton is a network of connected filaments and tubules extending from the nucleus to the plasma membrane. Dynamic: dismantles in one spot and reassembles in another to change cell shape It maintains the shape of the cell- fibers act like a geodesic dome to stabilize and balance opposing forces. Anchor organelles Move the cell and control internal movement of structures.
- 3. Motility of cells is determined by special organelles for locomotion. Internal movements (cytoplasmic streaming or cyclosis) by cytoskeleton components.
- 5. The ACTIN cytoskeletal machinery (red) is responsible for maintaining cell shape and generating force for movements. Polymerization and depolymerization of actin filaments (1) drives the membrane forward, whereas actin cross-linking proteins organize bundles and networks of filaments (2) that support overall cell shape. Movements within the cell and contractions at the cell membrane (3) are produced by myosin motor proteins. The actin (red) and intermediate filament (purple) cytoskeletons integrate a cell and its contents with other cells in tissues (4) through attachments to cell adhesions. Another type of intermediate filament, the nuclear lamins (5) are responsible for maintaining the structure of the nucleus.
- 6. Mechanical properties of Networks composed of actin, tubulin, and microtubules, actin filaments, or an intermediate filament called intermediate vimentin, all at equal concentration, filament polymers. were exposed to a shear force in a viscometer, & the resulting degree of stretch was measured. The results show that microtubule networks are strong, rigid hollow tubes that are easily deformed but rupture (red starburst) and begin to flow without limit when stretched beyond 150% of their original length. Actin filament networks are much more rigid, but they also rupture easily. Intermediate filament networks, by contrast, are not only easily deformed, but they withstand large stresses and strains without rupture; they are thereby well suited to maintain cell integrity.
- 7. Actin is encoded by a large, highly conserved gene family. Humans have 6 actin genes, which encode isoforms of the protein. Although differences among isoforms seem minor, the isoforms have different functions: α- actin is associated with contractile structures; γ-actin accounts for filaments in stress fibers; and ß-actin is at the front, or leading edge, of moving cells where actin filaments polymerize. Sequencing of actins from different sources has revealed that they are among the most conserved proteins in a cell, comparable with histones, the structural proteins of chromatin.
- 8. They form a dense complex web under the cell membrane. In intestinal cell microvilli, they act to shorten the cell In plant cells, actin filaments form tracts along which chloroplasts circulate. Involved in cell rigidity, tensile strength and resilience, cellular movement (pseudopodia and mesenchyme cell migration, platelet activation) Pseudopodia are associated with actin near the moving edge of the cell. Actin filaments move by interacting with myosin changing the configuration to pull the actin filament forward. Similar action accounts for pinching off cells during cell division and for amoeboid movement. Other arrangements of microfilaments in association with accessory proteins are possible. Ex: contractile rings of cell division; parallel bundles are found in stress fibers of fibroblasts, filopodia and other cell projections; gels of short randomly oriented filaments are found in egg cortical regions.
- 9. ACTIN AT THE CROSS ROADS
- 10. All cytoskeleton types form as helical assemblies of subunits that self-associate using a combination of end-to- end and side-to-side protein contacts. They grow fastest from the plus end than the minus end of the assembly. F-actin is polymerized through addition of globular actin or G-actin monomers at the growing (+) end, bearing a stabilizing ATP cap.
- 11. Nucleation of new actin filaments (red) is mediated by ARP complexes (orange) at the front of the web. Newly formed filaments are thereby attached to the sides of preexisting filaments. As these elongate, they push the plasma membrane forward. The actin filament plus ends will become protected by capping proteins (blue), preventing further assembly or disassembly from the old plus ends at the front of the array. Hydrolysis of ATP bound to the polymerized actin subunits promotes depolymerization at the rear end of the actin complex by a depolymerizing protein (green). The spatial separation of assembly and disassembly allows the network as a whole to move forward at a steady rate.
- 12. Treadmilling. Actin subunits can flow through the filaments by attaching preferentially to the (+) end and dissociating preferentially from the (-) end of the filament. Removal of monomers at the (–) end and addition of monomers at the (+) end leaves the filaments at the same overall length. The oldest subunits in a treadmilling filament lie at the (-) end. Treadmilling occurs at intermediate concentrations of free subunits.
- 13. Microfilaments in a cell. A crawling cell with 3 areas showing the arrangement of actin filaments. The actin filaments are shown in red, with arrowheads pointing toward the plus end. Stress fibers are contractile and exert tension. Filopodia are spike-like projections of the plasma membrane that allow a cell to explore its environments. The cortex underlies the plasma membrane.
- 14. A model of how forces generated in the actin-rich cortex move a cell forward. The actin-polymerization- dependent protrusion and firm attachment of a lamellipodium at the leading edge of the cell moves the edge forward (green arrows at front) and stretches the actin cortex. Contraction at the rear of the cell propels the body of the cell forward (green arrow at back) to relax some of the tension (traction). New focal contacts are made at the front, and old ones are disassembled at the back as the cell crawls forward. The same cycle can be repeated, moving the cell forward in a stepwise fashion. Alternatively, all steps can be tightly coordinated, moving the cell forward smoothly. The newly polymerized cortical actin is shown in red.
- 15. Platelet activation. (A) Platelet activation is a controlled sequence of actin filament severing, uncapping, elongation, recapping, and cross-linking that creates a dramatic shape change in the platelet. (B) SEM of platelets prior to activation. (C) An activated platelet with its large spread lamellipodium. (D) An activated platelet at a later stage than the one shown in C, after myosin II-mediated contraction.
- 16. Several actin-binding proteins influence deployment of filaments in the cytoplasm: Profilin binds to G-actin monomers to regulate polymerization Capping protein limits length increase by binding to the end of actin filament Fimbrin binds adjacent actin filaments to form bundles Filamin stabilizes filament 3-D network by intersecting with microfilaments Gelsolin breaks filament into shorter segments by inserting between subunits Vinculin & actinin mediate binding of actin to cell membrane at intercellular junctions and cell base.
- 18. The modular structures of four actin-cross-linking proteins Each of the proteins shown has two actin-binding sites (red) that are related in sequence. Fimbrin has two directly adjacent actin-binding sites, so that it holds its two actin filaments very close together (14 nm apart), aligned with the same polarity. The two actin-binding sites in α-actinin are separated by a spacer around 30 nm long, so that it forms more loosely packed actin bundles. Filamin has two actin-binding sites with a Vshaped linkage between them, so that it cross-links actin filaments into a network with the filaments oriented almost at right angles to one another. Spectrin is a tetramer of two α and two ß subunits, and the tetramer has two actin- binding sites spaced about 200 nm apart
- 19. Twisting of an actin filament induced by cofilin. (A) Three dimensional reconstruction from cryo-EM of filaments made of pure actin. The bracket shows the span of two turns of the actin helix. (B) Reconstruction of an actin filament coated with cofilin, which binds in a 1:1stoichiometry to actin subunits all along the filament. Cofilin is a small protein (14 kilodaltons) compared to actin (43 kilodaltons), and so the filament appears only slightly thicker. The energy of cofilin binding serves to deform the actin filament lattice, twisting it more tightly so that the distance spanned by two turns of the helix is reduced.
- 20. Filamin cross-links actin filaments into a three-dimensional network with the physical properties of a gel (A) Each filamin homodimer is about 160 nm long when fully extended and forms a flexible, high-angle link between two adjacent actin filaments. (B) A set of actin filaments cross-linked by filamin forms a mechanically strong web or gel.
- 21. INTERMEDIATE FILAMENTS Are structurally similar but biochemically distinct, with diameters intermediate between microtubules and microfilaments (about 10 nm). They associate with polypeptides fillagrin (binds to keratin), plectin (links vimentin), and synamin (also links vimentin, but found in muscle). 5 types are: 1. Glial filaments – found in non-neural cells of the CNS: astrocytes, oligodendrocytes, microglia. 2. Keratin filaments – characteristic of epithelial cells; called tonofilaments are often associated with desmosomes at the cell surface. They participate in the formation of keratin in keratinizing epithelia.
- 22. 3.Desmin – characteristic of smooth, striated & cardiac muscle; keep sarcomeres of neighboring myofibrils in register across the width of the fiber; link Z-bands of peripheral myofibrils to the sarcolemma; ensures uniform distribution of tensile strength throughout the muscle cell. 4.Vimentin – abundant in fibroblasts and mesenchymal derivatives, in bundles or randomly oriented in a network throughout the cytoplasm. 5.Neurofilaments – present in nerve cell processes with a cytoskeletal function; helps to maintain the gel state of the axoplasm; involved in intracellular metabolite transport.
- 23. A model of intermediate filament construction The monomer shown in (A) pairs with an identical monomer to form a dimer (B) in which the conserved central rod domains are aligned in parallel and wound together into a coiled coil. (C) Two dimers then line up side by side to form the tetramer soluble subunit of intermediate filaments. (D) Within each tetramer, the 2 dimers are offset with respect to one another, thereby allowing it to associate with another tetramer. (E) In the final 10-nm rope-like filament, tetramers are packed together in a helical array, which has 16 dimers in cross-section. Half of these dimers are pointing in each direction.
- 24. Keratin filaments in epithelial cells • Immunofluorescence micrograph of the network of keratin filaments (green) in a sheet of epithelial cells in culture. • The filaments in each cell are indirectly connected to those of its neighbors by desmosomes. • A 2nd protein (blue) has been stained to reveal the location of the cell boundaries.
- 25. Blistering of the skin caused by a mutant keratin gene. A mutant gene encoding a keratin protein was expressed in a transgenic mouse. The defective protein assembles with the normal keratins and thereby disrupts the keratin filament network in the basal cells of the skin. LM of cross sections of normal (A) and mutant (B) skin show that the blistering results from the rupturing of cells in the basal layer of the mutant epidermis (small red arrows). (C) Cells in the basal layer of the mutant epidermis, as observed by EM. As indicated by the red arrow, the cells rupture between the nucleus & the hemidesmosomes, which connect the keratin filaments to the underlying basal lamina.
- 26. Two types of intermediate filaments in cells of the nervous system. (A) Freeze-etch EM image of neurofilaments in a nerve cell axon, showing the extensive cross-linking through protein cross-bridges an arrangement believed to give this long cell process great tensile strength. The cross- bridges are formed by the long, nonhelical extensions at the C-terminus of the largest neurofilament protein (NF-H). (B) Freeze-etch image of glial filaments in glial cells, showing that these intermediate filaments are smooth and have few cross-bridges. (C) Conventional EM of a cross section of an axon showing the regular side-to-side spacing of the neurofilaments, which greatly outnumber the microtubules.