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Lecture 7

lecture 7 and 8.pdf

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Doug Thomson

Lecture 7 and 8: the cytoskeleton - Composed of 3 diff filament classes. Dynamic structure found in eukaryotes. o Provides structural transport, positions organelles, directs vesicular transport, involved in locomotion (flagella (sperm), cilia (paramecium), cell crawling (sheets of membrane crawl to active site), required for cell division, involved in muscle contraction o Proteins are directed to specific place b/w cytoskeleton - The cytoskeleton provides structural support: RBC poked with laser tweezers returns to its original shape due to structural support provided by cytoskeleton. It withstands tears and allows them to bring back to original shape - Video: - 3 types of filaments form the cytoskeleton: o Microfilaments: actin proteins. Diameter = 5-9nm. Form long filaments made of protofilaments that are twisted around each other o Intermediate filaments: intermediate filament proteins. Diameter = 10nm. They are flexible and extensive. Has rope-like qualities o Microtubules: made of turbulent proteins. Diameter = 25nm. Individual protofilaments allow structure to be organized to microtubule organised centre. - Techniques used to study cytoskeleton: o Immunoflorescence: technique used to determine the location of proteins within cell. Cells are FIXED. It is possible to locate more than one protein at a time and compare them o Antibody is used which binds specifically to protein of interest o Second antibody binds to the first antibody and is covalently tagged with fluorescent molecule o Fluorescence microscope is used to excite fluorescence molecule and visualise light emitted - Light microscopy has resolution limit due to diffraction based on wavelength of light (250 nm) - Electron microscope: electrons, shorter wavelength. Resolution: <10 Angstroms (>250x better resolution) - Dynamic structure: electron microscope is not useful for this. o For each cell motility/crawling the actin filaments must rapidly assemble and disassemble at the leading edge  Eg. a neutrophil chasing clump of bacteria  Eg. Microtubules: most interphase microtubules radiate from one microtubule organizing center which is located near nucleus.  they are recognized to form bipolar mitotic spindles in dividing cells - cytoskeletal filaments are composed of small soluble subunits that form polymers which allows them to be dynamic. Can rapidly assemble and disassemble - How are these polymers constructed: need to be strong so that they can have support functions within cell. They need to be flexible and easily disassemble and reassemble. o Cytoskeletal filaments constructed of multiple chains Single chain polymers with: Strong bonds Weak bonds Thermal stability High Low Dynamic filament Low High - Multiple subunits are assembled into bundles of protofilaments: individual protofilaments associate with each other laterally o Strength of cytoskeletal filments is due to many weak bonds. Together they build a stronger filaments o Each individual bond doesn’t need to be as strong and that allows the cyctoskeletal components to be both strong and dynamic. o All actin filaments, intermediate filaments, and microtubules are all constructed of more than one protofilament - Actin polymerisation in vitro experiments: high concentration of actin subunits is added to the test tube under low salt conditions. When salt added it helps favour polymerase reaction (subunits start to form up into filaments). There is a lag time after addition of salt (during this step an association b/w 2 subunits might form that eventually break apart). Nucleus formed (association of 3 or more subunits), here bonds start to come stabilized and nucleus is at a position where more subunits can be incorporated. Actin filaments start to grow = linear growth by addition of proteins from soluble pool. Once filaments get to certain size, reaches equilibrium where the net addition of soluble pool = loss of filament. There is no growth of filament after this (critical concentration: concentration of free filament subunits at which the filaments in solution will be at a steady state with soluble pool). - Microtubules involved in: intracellular transport (radiate out from center of cell have motor proteins that move system giving them directionality), structural support, cell organization (position organelles), mitosis (by forming spindle), cell motility (flagella and cilia) - Microtubules made of: tubulin, long hollow tubes, stiff (like straw), inextensible (don’t extend) o Made of individual subunits of alpha and beta tubulin that form hetrodimer o Tubulin heterodimer = microtubule subunit. Tubule contains of 13 proteins. o Two closely related globular proteins form tubulin heterodimier o This arrangement of alpha and beta subunits give the microtubule polarity. It has a plus end (beta) and a minus end (alpha)  The minus ends are organized to the centre of the cell attached to the centrosome - 13 parallel protofilaments make up hollow tubule. All arranged in same orientation o All bonds b/w individual subunits are non covalent o The bonds b/w protofilaments are weaker than the bonds w/in each protofilament o Growth and disassembly of microtubules occurs at ends - In vitro microtubule growth is faster at the plus end o Isolated microtubule structure (eg. cilia) are incubated w/ a high concentration of tubulin and GTP o This bundle of microtubules isolated form a cilium o Growth is much faster in vitro in plus end compared to the minus end - Tubulin dimers: free dimers are bound to GTP (guanosine triphosphate) called T-Form o Tubulin subunits are enzymes and they are able to hydrolyze GTP o When this occurs in the filament GDP is trapped in tubulin subunits (D-Form). D-Form is tubulin is bound to GDP – happens only in filaments. In filament there is hydrolysis of GTP and over time subunits move to D Form. - How these 2 forms affect assembly of microtubules: soluble subunits are in T Form o When you got polymerization incorporated into filament as T form. Over time subunits in filament can hydrolyze GTP which traps GDP and they become D Form. Because addition is faster at plus end (GTP added) plus end will have more T form dimers. Minus end have more of D form dimers. o Microtubules have a GTP cap at the plus end when: dimer addition at the plus end is faster than GTP hydrolysis. GTP cap helps stabilize plus end of microtubule. - The microtubule GTP cap stabilizes the plus end o GTP cap is at the plus end (faster growing end). o GTP cap stabilizes the plus end favours tubule grow
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