Biology 2382B Lecture Notes - Proline, A Band, Actomyosin Ring

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Published on 17 Apr 2013
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Section 1
Microtubules
(pg. 757-791)
18.1 Microtubule Structure and Organization
-mitotic spindle
-flagella/cilia
-components of axons
oMade of 13 longitudinal repeating units called protofilaments
oBrain microtubules were purified to find a protein called tubulin, which is a Microtubule
Associated Protein (MAP)
Microtubule Walls are Polarized Structures Built from αβ- Tubulin
-Purified soluble tubulin is a dimer made of two closely related subunits; α and β tubulin of 55000
Daltons each (there is also γ-tubulin, which is used in regulatory function)
-Each subunit of the tubulin dimer can bind one molecule of GTP
-GTP trapped in the α-tubulin is never hydrolyzed
-GTP in the β-tubulin is exchangeable with the free GTP, and can be hydrolyzed
-MT consist of 13 laterally associated protofilaments forming a tube whose external diameter is 25nm
-Each protofilaments is made up of alternating αβ-tubulin dimers, with each subunit type repeating each
8nm
-Each protofilaments has an alpha at one end and a beta at the other end, giving the protofilaments an
intrinsic polarity
-MT have an overall polarity, one end is (+) and one is (-)
-The (+) (fast) end is favoured by polymerization with exposed β subunits
-The (-) (slow) end has exposed α subunits
-Most MT in a cell consist of a simple tube, a
singlet MT, made of 13 protofilaments (rare
cases where a singlet contains fewer or more
protofilaments)
-Doublet & Triplet MT are found in specialized
structures such as flagella and cilia (doublet)
and centrioles and basal bodies (triplet)
-Each doublet/triplet contains a 13-
protofilament MT (A tubule) and one/two
additional tubules (B and C) consisting of 10 protofilaments each
Microtubules Are Assembled from MTOC’s to Generate Diverse Organizations
-all MT are nucleated from structures known as Microtubule-organizing centres; MTOCs
-In most cases the (-) end of the MT stays anchored in the MTOC
-MTOC in interphase is the centrosome and is generally near the nucleus, producing a radial array of
MT with the (+) ends toward the cell edges
-This provides tracks for MT-based motor proteins to organize and transport membrane bound
compartments, such as those comprising the secretory and endocytic pathways
-MITOSIS: cells completely reorganize their MT to form a bipolar spindle, assembled from two
MTOCs called the spindle poles
-Neurons have long processes called axons, in which organelles are passed in both directions along MT.
The MT in the axons are not continuous but have been released from the MTOC, but all have the same
polarity
-The MT in dendrites have mixed polarity (Reason unknown)
-Flagella and cilia MT are from an MTOC called a basal body
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-Centrosomes in animal cells consist of a pair of orthogonally arranged cylindrical centrioles
surrounded by pericentriolar material
-Centrioles which are about 0.5μm long and 0.2μm in diameter, are highly organized and stable
structures that consist of nine sets of triplet MT and are closely related in structure to basal bodies
-It’s not the centrioles themselves that nucleate the cytoplasmic MT array, but factors in the
pericentriolar material >
-γ-tubulin ring complex – this protein complex is located in the pericentriolar material and consists of
many copies of γ-tubulin, it is believed γ – TURC acts like a split washer template to bind αβ tubulin
dimers
-Centrisomes also anchor and regulate the (-) end of MT
-Basal bodies have a similar MTOC at the base of cilia/flagella, the A&B tubules of their (mtoc) triplet
MT provide a template for the assembly of the MT that make of the core structure of the cilia and
flagella
18.2 Microtubule Dynamics
-MT lifespan during mitosis: 1 minute
-MT non-mitotic lifespan: 5-10 minutes
-Microtubule lifespan is longer in axons and much longer in flagella and cilia
Microtubules Are Dynamic Structures Due to Kinetic Differences at Their Ends
-MAPs catalyze MT (de)polymerization
-Disassembly of MT occurs at 4ºC and reassemble into MT again at 37ºC
-A slow nucleation phase, followed by a rapid elongation phase and then a steady state phase in which
assembly is balanced by disassembly
-For assembly to occur, the αβ tubulin concentration needs to be above the critical concentration (Cc);
when αβ is above the Cc, then polymerization occurs – and when it’s below Cc, the MT depolymerise
-The higher the concentration above the Cc, the faster they MT polymerizes
-When the concentration is below a certain point, there is tread-milling
Dynamic Instability
-MT undergo dynamic instability, meaning that they undergo periods of growth followed by periods of
shrinkage
-A growing MT has a blunt end, whereas depolymerising end has a frayed end
-Growing MT with blunt ends terminate in GTP β tubulin, whereas shrinking ones with frayed ends
terminate in GDP β tubulin.
-Therefore, if the GTP in the terminal β tubulin of a MT become hydrolyzed, a formerly blunt-end
growing MT will curl and fray
-Growing MT are capped by GTP-β tubulin
Drugs Affecting Tubulin
-Colchicine causes depolymerisation of MT (binds to tubulin diamers so that they can’t polymerize) –
joint pain of acute gout, relieves inflammation caused in gout by reducing the MT dynamics of WBC,
rendering them unable to migrate efficiently to the site of inflammation
-Taxol provides stability to growing MT, taxol stops cells from dividing by inhibiting mitosis, it has
been used to treat come cancers (ie. Breast and ovary- these cells are very sensitive to the drub)
Regulation of Microtubule Structure and Dynamics
-MT are stabilized by side and end binding proteins called MAPs
-There are many different proteins that stabilize MT, many of them show cell specificity.
-Best studied are the tau family of proteins which includes; tau, MAP2 and MAP4
-These proteins have a modular design, with 18-residue positively charged sequences, repeated three to
four times, that binds to the negatively charged tubulin surface and a domain that projects from the MT
wall.
-Tau proteins are believed to stabilize MT and also act as spacers between them
-MAP2 is found only in dendrites, where it forms fibrous cross-bridges between MT and links MT to
intermediate filaments, IF
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-Tau is present in both axons and dendrites
-In many cases the activity of the MAPs is regulated by the reversible phosphorylation of their
projection domain
-Phosphorylated MAPs are unable to bind to MT; thus phosphorylation promotes depolymerisation
-Microtubule-affinity-regulating kinase (MARK/Par-1) is a key modulator of tau proteins
-Some MAPs are also phosphorylated by a cyclin-dependant kinase (CDK) that plays a major role in
controlling the activities of proteins in the course of the cell cycle
-Some MAPs associated with the (+) end of MT, and in some cases only the (+) ends of growing MT
-This class of proteins are known as +TIPs, and they perform varied functions when present at the MT
tip
-Some selectively stabilize the (+) end against shrinkage or enhance the frequency of rescues (promote
continuous growth)
-Other +TIPs are associated with proteins used for attachment to an organelle or cell wall (migrating
cell leading edge)
-One specific +TIP is EB1, which is associated with the (+) end of the MT as well as the seam; location
is known, function is not entirely know; some package and move etc etc
Section 2
Microtubules are disassembled by End Binding and Severing Proteins
-binding kinesin-13 to remove dimers from the (+) end which will cause depolymerisation, ATP usage
is needed for this reaction
-Stathmin binds tubulin dimers in the curve and may also cause GTP hydrolysis, removing the cap at
the (+) which will induce shrinkage it has been found that phosphorylation near the leading edge of
motile cells inactivates Stathmin/Op18
-Katanin severs and releases anchored MTs and this also removes the cap at the (+) end which will
allow for depolymerization to occur
Microtubules: tracks for transport
-Vesicle transport is bi-directional
-Motor proteins doing the movement and they need ATP in order to move anything
-Axonal transport: observed from a squid giant axon, uses ATP to move things up and down the MT
Kinesin-1 Powers Anterograde Transport of Vesicles
Down Axons Toward the (+) end of MT
-14 known classes (45 genes in humans)
-2 heavy chains – head, flexible neck and stalk
-2 light chains (variable)
-Heavy chain heads have ATPase activity and MT
binding ability
-Light chains recognize cargo
-Most are (+) directed
-The head binds to the MT and uses ATP to “walk”
along the MT
-The light chain recognizes cargo
-
-Kinesin 1&2, work by walking towards the (+) end,
pulling a type of “cargo” (ie. Organelles)
-Kinesin 5: works between MT, heads on both ends
and walk in opposite direction causing a sliding
-Kinesin 13: works in the disassembly of MT by
removing dimers from the (+) OR (-) end
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