Lecture 7 and 8.docx

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14 Apr 2012
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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)
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