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Biological Sciences
Rene Harrison

Cytoskeleton 1. What are cytosolic proteins and what is their function?  Cytosolic proteins don‟t have a transfer sequence since they just stay in the cytoplasm.  The cytosol is a fluid matrix surrounding organelles and contains water, ions, sugars, ATP, free ribosomes and many proteins 2. What is the targeting sequence for proteins destined to stay in cytosol?  The targeting sequence for proteins destined to stay in the cytosol is nothing; there is no targeting sequence for these proteins.  Proteins are translated in the cytosol and stay there by “default” 3. What are some types of cytosolic proteins that can be found?  Examples of two types of cytosolic proteins that can be found are: Rabs and SNARES, coat proteins like the clathrin and cops, there is also signaling proteins – kinases, phosphatases, GTPases, glycolysis enzymes organelle proteins “in transit”, and chaperone proteins, soluble receptors for transport organelle proteins (example Hsp70), and lastly cytoskeleton 4. What is the cytoskeleton made up of?  The cytoskeleton is made up of 3 components: a. Microtubules b. Actin c. Intermediate filaments  The cytoskeleton is made of an extensive protein networks and cytosolic proteins, and are made on free ribosomes with no targeting sequence 5. What is the function of the cytoskeleton?  The function of the cytoskeleton is for: a. Dynamic scaffold – structure and support b. Spatial positioning of organelles c. Intracellular transport d. Cell contraction/Cell motility/Phagocytosis e. Machinery for cell division Figure 9.1 – An overview of the structure and (a) an epithelial cell, (b) a nerve cell, and (c) a dividing cell. The microtubules of the epithelial and nerve cells function primarily in support and organelle transport, whereas the microtubules of the dividing cell form the mitotic spindle required for chromosome segregation. Intermediate filaments provide structural support for both the epithelial cell and nerve cell. Microfilaments support the microvilli of the epithelial cell and are an integral part of the motile machinery involved in nerve cell elongation and cell division Figure 9.2 – An example of the role of microtubules in transporting organelles. The peroxisomes of this cell are closely associated with microtubules of the cytoskeleton. Peroxisomes appear green because they contain a peroxisomal protein fused to the green fluorescent protein. Microtubules appear red because they are stained with a fluorescently labeled antibody. Figure 9.11 – Localization of microtubules of a flattened, cultured anti-tubulin antibodies. Microtubules are seen to extend from a perinuclear region of the cell in a radial array. Individual microtubules can be followed and are seen to curve gradually as they conform to the shape of the cell 6. What are microtubules (MTs)?  Microtubules are composed of tubulin heterodimers each with an a- and b-tubulin subunits which polymerize to make MTs  Heterodimers arrange into protofilaments  Microtubules are tertiary structures from what can be seen in x-ray crystallography Figure 9.9 – The structure of microtubules. Diagram of a longitudinal section of a microtubule shown in the B-lattice, which is the structure thought to occur in the cell. The wall consists of 13 profilaments composed of aB-tubulin heterodimers stacked in a head-to-tail arrangement. Adjacent profilaments are not aligned in register but are staggered about 1nm so that the tubulin molecules form a helical array around the circumference of the microtubule. The helix is interrupted at one site where a and B subunits make lateral contacts. This produces a “seam” that runs the length of the microtubule. 7. What is the function of microtubules (MTs)?  Tubulin heterodimers – B-tubulin binds GTP to allow polymerization and the GTP slowly converts to GDP after incorporation into the MT wall  This polymerizes into a protofilament which is a 13 protofilaments arranged around a hallow core and forms the microtubule (MT)  MTs have polarity thus with a plus and minus end  The ends structurally alittle different the negative end is embedded in the centrosome which is the MT-organizing centre (MTOC) where polymerization begins  This starts with gamma-tubulin and slightly different structure than the a and B tubulin  The positive ends extend towards the plasma membrane  MTs polymerize and depolymerize at the positive end called the growth and shrinkage Figure 9.9 – The structure of microtubules. EM of a cross section through a microtubule of a juniperus root tip cell revealing 13 subunits arranged within the wall of the tubule. Figure 9.13 – Axonal transport. EMof a portion of axon from a cultured nerve cell, showing the numerous parallel microtubules that function as tracks for axonal transport Figure 9.18 – The centrosome. Fluorescence micrograph of cultured mammalian cell showing the centrosome at the center of an extensive microtubular network Figure 9.8 – The study of the cytoskeleton using FRAP. Micrographs from a FRAP experiment performed on interphase cells expressing GFP tubulin. Two side-by-side cells were bleached in the boxed region with a laser, and the cells were then imaged over time. 8. What does Figure 9.19 Microtubule nucleation at the centrosome signify?  In picture (a) Fluorescent micrograph of a cultured fibroblast that had been exposed to colcemid to bring about the disassembly of the cell‟s microtubules and was then allowed to recover for 30 minutes before treatment with fluorescent anti-tubulin antibodies. The bright starlike structure marks the centrosome together with newly assembled microtubules that have begun to grow outward in all directions.  (b) Schematic diagram of the growth of microtubules showing the addition of subunits at the plus end of the polymer away from the centrosome.  Thus basically in the experiment the biologists washed out the drug quickly to see if the microtubules polymerize and to where they came from: thus they removed the drug and watched MTs reassemble – assembled at the MTOC/centrosome 9. What else are MTs capable of doing?  MTs rapidly turnover, that is that they polymerize and depolymerize from end called “dynamic instability” which insists that MTs are constantly growing and shrinking  The half-life of average MT is 10 minutes and MTs stability can be increased (some are stable)  Also the binding of structural MAPs (MT-associated proteins) to the walls of MTs, eg. MAP2, tau can prevent MTs from disassembling and depolymerizing Figure 9.10 – Microtubule-associated proteins (MAPs). Schematic diagram of a brain MAP2 molecule bound to the figure contains three tubulin-binding sites connected by short segments of the polypeptide change. The binding sites are spaced at a sufficient distance to allow the MAP2 molecule to attach to three separate tubulin subunits in the wall of the microtubule. The tails of the MAP molecules project outward, allowing them to interact with other cellular components. - MT wall - important componentom falling apart and is a very - Stable MTs are frequently used to transport organelles and function as “highways” 10. What are microtubule motors?
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