Difference between revisions of "Cytoskeleton - Microtubules"

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==Lecture Content to be added==
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== '''Cytoskeleton - Microtubules''' ==
 +
[[Image:Microtubule1.jpg|thumb|Microtubules]]
 +
This lecture continues to look at the cell cytoskeleton, covering in detail the microtubule system.
 +
 
 +
[[Image:Myosin Microtubule Actin Collagen.jpg|thumb|Molecular sizes]]
 +
Microtubules are the largest filament system of the cytoskeleton and have important functions for intracellular motility of nearly all cytoplasmic structures (organelles, vesicles, and smaller components).
 +
 
 +
('''MH''' - Note that the role of microtubules in mitosis will not be covered in detail, as this topic is covered elsewhere in lecture series)
 +
 
 +
The key concepts are: microtubules, intracellular motility, tubulin, microtubule associated proteins, microtubule motors, centrosome, flagella, cilia The lecture slides and textbook alone contain enough information as an introduction to the subject for this level of study. If you are interested in further reading, I have also included below links to more detailed textbooks with further information and images. Please note this additional information is not necessarily examinable, but may be useful if you have not previously studied biology.
 +
 
 +
 
 +
 
 +
== Lecture Slides ==
 +
 
 +
'''2010''' Microtubule Lecture Slides [[media:Lecture11.pdf|Lecture11]]
 +
 
 +
== Objectives ==
 +
* Understand the structure and role of microtubules
 +
* Understand tubulin structure
 +
* Understand motor molecules of the microtubule network
 +
* Brief understanding of microtubule associated proteins
 +
* Brief understanding of drugs that effect microtubules
 +
* Brief understanding of microtubule role in mitosis
 +
 
 +
==Lecture Overview==
 +
* Microtubules (mt)
 +
* Structure
 +
* Formation
 +
* Function (Will not cover mitosis in detail today, see cell division lecture)
 +
* Polarity
 +
* Turnover
 +
* Associated Proteins (map)
 +
* Motors
 +
* Disorders - Alzheimers, cancer therapies
 +
 
 +
 
 +
==About Microtubules==
 +
* Cell organizing role
 +
* Cytoskeleton
 +
** Largest fibre
 +
** 25 nm diameter
 +
** cytoplasmic
 +
* All cells contain
 +
** Same core structure
 +
** Same motors
 +
** Different associated proteins
 +
* Dynamic
 +
** Continuous remodelling
 +
* Movement
 +
** Intracellular > cellular
 +
** Cell division
 +
 
 +
 
 +
== Motility- Intracellular ==
 +
 
 +
organelle movement
 +
vesicle transport
 +
mitosis & meiosis
 +
chromosome segregation
 +
gene expression
 +
transcription factor binding
 +
mRNA transport
 +
translation
 +
protein export
 +
transmitter release
 +
Motility- Intracellular
 +
Axon transported vesicles
 +
EM axonal transported vesicles and axonal cytoskeleton in longitudinal section
 +
Arrows show rod shaped structures
 +
appear as cross bridges between organelles and microtubules
 +
Scale bar 100 nm
 +
 
 +
 
 +
== Basal Bodies of Cilia ==
 +
 
 +
Surface of ependymal cell
 +
contains basal bodies
 +
red rings
 +
connected to cilia  microtubules
 +
longitudinal section
 +
Inset: cilia transverse section
 +
central MT doublet
 +
surrounded by nine MT pairs
 +
one of each pair has a hook-like appendage (arrows) Å~100,000
 +
see later motor slides
 +
 
 +
 
 +
== Endocytic Pathway ==
 +
 
 +
Endocytic movement
 +
occurs along microtubules
 +
can be blocked by drugs
 +
Depolymerizing drugs
 +
Stabilizing drugs
 +
 
 +
 
 +
== Fish Pigment Cell Model ==
 +
 
 +
Changes in skin coloration in fish
 +
Contain large pigment granules (brown)
 +
change location in response to neuronal or hormonal stimulus
 +
Dispersal and aggregation of pigment granules occur along MTs
 +
Movie: GFP-Tubulin
 +
Movie: Microtubules and
 Cell Division
 +
MT Mitotic Spindle
 +
MT Capture of Kinetochores
 +
Movie: MT and Yeast
 +
 
 +
 
 +
==Structure ==
 +
 
 +
Long hollow tubes
 +
25 nm diameter
 +
Radiate from forming structure
 +
Centrosome
 +
Spindle pole
 +
Basal Body
 +
Polarized
 +
(+) plus and (-) minus ends
 +
Formed from Tubulin
 +
55 kD protein
 +
 
 +
=== Microtubule Structure ===
 +
 
 +
(A) EM of  mt in cross-section
 +
ring of 13 distinct subunits
 +
Each a separate tubulin molecule
 +
an alpha/beta heterodimer
 +
(B) EM of a mt assembled in vitro
 +
(C) 13 molecules in cross-section
 +
(D) side view of a mt
 +
tubulin molecules aligned into long parallel rows
 +
13 Protofilaments
 +
Each is composed of a series of tubulin molecules, each an a/b heterodimer
 +
mt is a polar structure with a different end of tubulin molecule (a or b) facing each end of microtubule
 +
 
 +
===Tubulin Protofilaments===
 +
dimers polymerize to form microtubules
 +
13 linear protofilaments
 +
head-to-tail arrays of tubulin dimers
 +
arranged in parallel
 +
assembled around  hollow core
 +
 
 +
=== Microtubule Polarity ===
 +
 
 +
Tubulin subunits in a MT
 +
subunits aligned end to end into a protofilament
 +
magenta highlight
 +
side-by-side protofilament packing forms wall of microtubule
 +
slightly staggered so that a-tubulin in one protofilament contacts b-tubulin in neighboring protofilaments
 +
 
 +
===Arrangement of Protofilaments===
 +
 
 +
Singlet
 +
typical microtubule
 +
tube built from 13 protofilaments
 +
Doublet
 +
additional set of 10 protofilaments
 +
form a second tubule by fusing to the wall of a singlet
 +
Triplet
 +
Attachment of another 10 protofilaments
 +
 
 +
===Tubulin===
 +
* dimer 55-kd polypeptides
 +
** α-tubulin (alpha-)
 +
** β-tubulin (beta-)
 +
* encoded by related genes
 +
* third type of tubulin
 +
** γ-tubulin (gamma-)
 +
* located at centrosome
 +
* role in initiating mt assembly
 +
 
 +
===Tubulin Genes===
 +
* human DNA contains about 14 copies per genome of both genes Cleveland et al. (1980)
 +
** Beta β 6p21.3 - 15 to 20 members
 +
** Alpha α mainly Chr.12 - 15 to 20 dispersed genes
 +
** Gamma γ 17q21
 +
* Also many tubulin pseudogenes
 +
 
 +
===Tubulin Synthesis Regulation===
 +
 
 +
autoregulation in animal cells
 +
stability of polysome-bound tubulin mRNAs
 +
beta-tubulin RNAs
 +
selectively targeted as substrates for destabilization
 +
not recognition of specific RNA sequences
 +
co-translational recognition of amino-terminal beta-tubulin tetrapeptide after emergence from ribosome
 +
Motif could be used in other systems where RNA degradation is coupled to ribosome attachment and translation
 +
 
 +
===Tubulin Homology===
 +
 
 +
FtsZ
 +
bacterial GTPase (40,000 Mr)
 +
bacterial protein has structural and functional similarities with tubulin
 +
ability to polymerize and a role in cell division
 +
protein carrying out these ancestral functions in bacteria was modified during evolution to fulfill diverse roles of microtubules in eukaryotes?
 +
 
 +
 
 +
== Centrosome ==
 +
 
 +
slow-growing minus end of MT embedded in centrosome matrix surrounding a pair of centrioles
 +
matrix determines number of MTs in a cell
 +
By nucleating growth of new MTs
 +
Microtubule Organization
 +
Movie: Microtubules and Mitochondria
 +
Movie: Microtubules and Endoplasmic Reticulum
 +
 
 +
===Centrosome Cycle===
 +
 
 +
Orientation of MTs in Cells
 +
(-) Minus ends of MTs  generally embedded in a microtubule-organizing center (mtoc)
 +
alpha
 +
(+) plus ends often located near the plasma membrane
 +
beta
 +
 
 +
 
 +
=== Orientation of cellular MT ===
 +
 
 +
Neuron- Axon and Dendrites
 +
Gamma-Tubulin-mediated assembly of microtubules
 +
models for g-tubulin-mediated assembly of microtubules
 +
alternative models
 +
gamma -TuRC nucleates microtubule assembly either by
 +
left- presenting a row of gamma -tubulin subunits
 +
right- forming a protofilament, which can directly bind a/b-tubulin subunits
 +
 
 +
modified from C. Wiese and Y. Zheng, 1999, Curr. Opin. Struc. Biol. 9:250–259.
 +
 
 +
 
 +
== MT Dynamic Instability ==
 +
 
 +
continual and rapid MT turnover
 +
half-lives of only several minutes
 +
this rapid turnover critical for remodeling of the cytoskeleton during mitosis
 +
Tim Mitchison and Marc Kirschner (1984)
 +
Movie: GFP mt
 +
Movie: GFP mt
 +
Movie: GFP mt
 +
 
 +
===MT Treadmilling===
 +
 
 +
Treadmilling
 +
dynamic behavior when tubulin bound to GDP continually lost from minus end
 +
replaced by the addition of tubulin bound to GTP to plus end of same microtubule
 +
GTP hydrolysis also results in dynamic instability
 +
individual microtubules alternate between cycles of growth and shrinkage
 +
 
 +
 
 +
 
 +
 
 +
== Microtubule Movement ==
 +
 
 +
GTP hydrolysis destabilizes MTs
 +
Addition of tubulin adds GTP to end of  protofilament
 +
grows in linear conformation readily packed into MT wall
 +
becoming stabilized
 +
Hydrolysis of GTP
 +
changes subunits conformation
 +
force protofilament a curved shape
 +
less able to pack into the MT wall
 +
protofilaments with GDP-containing subunits forced linear conformation by lateral bonds within MT wall, mainly in stable cap of GTP-containing subunits
 +
 
 +
 
 +
== GTP Cap Hydrolysis ==
 +
 
 +
GTP hydrolysis
 +
destabilizes MTs
 +
GDP-containing protofilaments relax
 +
curved conformation
 +
progressive disruption of MT
 +
disassembly of protofilaments
 +
free tubulin dimers
 +
 
 +
 
 +
== MT Stability and cell Polarity ==
 +
 
 +
Microtubule Associated Proteins
 +
Many different proteins
 +
Example - neurons
 +
 
 +
 
 +
== Microtubule Associated Proteins ==
 +
 
 +
===Microtubule Associated Protein===
 +
* MAP2
 +
* Neuron expression
 +
* a 280-kD protein
 +
* concentrated in neuronal soma and dendrites
 +
* Developmentally regulated expression
 +
* 2q34-q35
 +
* MAP2 Developmental Expression
 +
** MAP2B  - present throughout brain development
 +
** MAP2A - appears during end of second week of  postnatal life
 +
** MAP2C - present during early brain development, disappears from the mature brain, except for the retina, olfactory bulb, and cerebellum
 +
* MAP2A and MAP2B
 +
** encoded by 9-kb mRNAs
 +
* MAP2C
 +
** encoded by a 6-kb mRNA
 +
===Tau===
 +
* Protein Mr 45-60 kDa
 +
* Gene 17q21.1
 +
Neuron Expression
 +
Enriched in axons
 +
phosphorylated
 +
Tau- Alzheimer Disease
 +
neuronal cytoskeleton is progressively disrupted
 +
replaced by tangles of paired helical filaments (PHFs)
 +
PHFs composed mainly of hyperphosphorylated form of Tau
 +
Tau- Alzheimer Disease
 +
Elevated tau inhibit intracellular transport
 +
mainly plus-directed transport (kinesin motors)
 +
from center of cell body to neuronal processes
 +
organelles are unable to penetrate neurites
 +
peroxisomes, mitochondria, and transport vesicles carrying supplies for growth cone
 +
Leads to
 +
stunted growth
 +
increased susceptibility to oxidative stress
 +
pathologic aggregation of proteins such as amyloid precursor protein (APP)
 +
tau:tubulin ratio is normally low
 +
increased levels of tau become detrimental to the cell
 +
 
 +
 
 +
== Microtubule Motors ==
 +
 
 +
 
 +
=== Microtubule Motor Proteins ===
 +
 
 +
* Dynein (-) and Kinesin (+) move in opposite directions
 +
* globular heads of heavy chains bind mts
 +
* motor domains
 +
 
 +
==== Dynein ====
 +
* - (minus) end motor
 +
* 2 or 3 heavy chains (two are shown here)
 +
* multiple light and intermediate chains
 +
 
 +
====Kinesin====
 +
* + (plus) end motor
 +
* 2 heavy chains, wound around each other in a coiled-coil structure
 +
* 2 light chains
 +
* Movie: Kinesin on Microtubule
 +
* Movie: Microtubules in vitro
 +
 
 +
===Ciliary and Flagellar Axonemes===
 +
* 9 + 2 MT arrangement
 +
* dynein arms and radial spokes with attached heads occur at intervals along the longitudinal axis
 +
* central microtubules, C1 and C2
 +
* Axonemal Dynein
 +
** Arrangement of globular domains and short stalks
 +
** attachment of outer dynein arm to the A tubule of one doublet and cross-bridges to B tubule of an adjacent doublet
 +
** attachment to A tubule is stable
 +
Presence of ATP
 +
successive formation and breakage of cross-bridges to adjacent B tubule
 +
leads to movement of one doublet relative to the other
 +
Dynein-mediated sliding of axonemal mt
 +
Dynein arm attach to A subfiber of one microtubule
 +
walk along B subfiber of adjacent doublet
 +
toward (-) end (small arrow)
 +
moves microtubule in opposite direction (large arrow)
 +
cross-links (nexin) broken, sliding can continue
 +
 
 +
 
 +
==Microtublues In Development==
 +
* nurse cells in insect ovarioles
 +
* supply oocytes cellular components
 +
* mRNAs, proteins
 +
* pass from one cell to another through intercellular bridges traversed by microtubules
 +
* mRNAs encode axis-determining factors in Drosophila embryos
 +
* mRNAs are translocated
 +
* localized within oocyte
 +
* sites where translation products will function
 +
 
 +
 
 +
== Microtubule Drugs and Cancer ==
 +
 
 +
* drugs affect microtubule assembly
 +
* experimental cell biology tool
 +
* Cancer treatment
 +
* Colchicine and Colcemid
 +
** bind tubulin
 +
** inhibit mt polymerization, blocks mitosis
 +
* Vincristine and Vinblastine
 +
** cancer chemotherapy
 +
** selectively inhibit rapidly dividing cells
 +
* Taxol
 +
** stabilizes microtubules rather than inhibiting their assembly
 +
** also blocks cell division
 +
===Taxol - Paclitaxel===
 +
* 1971 from the bark of the Western yew
 +
* Taxus brevifolia Nut (Taxaceae)
 +
* Anti-tumor and anti-leukemic activity
 +
* found in roots, leaves, and stems of this and related members of yew family
 +
* complex ester
 +
* an oxetan ring attached to a derivative of taxane
 +
* tool for investigating MT function
 +
* clinical trials in a variety of cancers
 +
* Initial development limited by low abundance in yew trees
 +
* now novel synthetic methods
 +
* identification of new sources of taxanes
 +
 
 +
 
 +
== References ==
 +
 
 +
===Textbooks===
 +
 
 +
====Essential Cell Biology====
 +
*  Chapter 16, p518-527
 +
 
 +
====Molecular Biology of the Cell====
 +
Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter
 +
New York and London: Garland Science; c2002
 +
* Molecular Biology of the Cell Ch16, p803-820
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=cell.section.4222 microtubules]
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=cell.figgrp.4190 Image: growth and shrinkage of microtubules]
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=cell.section.4257 Cilia and Centrioles]
 +
* [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=microfilament&rid=mboc4.table.2992 Table 16-2.  Drugs That Affect Actin Filaments and Microtubules]
 +
 
 +
====Molecular Cell Biology====
 +
Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E.
 +
New York: W. H. Freeman & Co.; c1999
 +
* Molecular Cell Biology Chapter 19
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mcb.section.5406 microtubule structures]
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mcb.section.5428 Microtubule Dynamics and Associated Proteins]
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mcb.section.5452 Kinesin, Dynein, and Intracellular Transport]
 +
 
 +
====The Cell- A Molecular Approach====
 +
Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000
 +
* The Cell Chapter 8
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=cooper.section.1820 microtubules]
 +
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=cooper.figgrp.1825 Image: Intracellular organization of microtubules]
 +
 
 +
====Search Online Textbooks====
 +
 
 +
* "microtubule" [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=microtubule+mboc4 Molecular Biology of the Cell] | [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=microtubule+mcb Molecular Cell Biology] | [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=microtubule+cooper The Cell- A molecular Approach] | [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=microtubule+filament Bookshelf]
 +
 
 +
===Books===
 +
* [http://books.google.com/books?id=6OKS_xx9xTsC Cell Junctions: Adhesion, Development, and Disease‎ by Susan E. La Flamme, Andrew Kowalczyk]
 +
 
 +
===PubMed===
 +
* '''PubMed''' is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed PubMed]
 +
 
 +
* '''PubMed Central''' (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=PMC PMC]
 +
 
 +
* '''Online Mendelian Inheritance in Man''' (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes.  [http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim OMIM]
 +
 
 +
* '''Entrez''' is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others [http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar Entrez]
 +
 
 +
====Search Pubmed====
 +
2007 Medline search “microtubule” 34899 References, 3523 Reviews
 +
* "microtubule"  [http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar&cmd=search&term=microtubule Entrez all databases]
 +
* "tubulin"  [http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar&cmd=search&term=tubulin Entrez all databases]
 +
* "microtubule+motor"  [http://www.ncbi.nlm.nih.gov/sites/gquery?itool=toolbar&cmd=search&term=microtubule+motor Entrez all databases]
 +
 
 +
====Reviews====
 +
 
 +
* Structure and function of mammalian cilia. Satir P, Christensen ST. Histochem Cell Biol. 2008 Jun;129(6):687-93. Epub 2008 Mar 26. Review. [http://www.ncbi.nlm.nih.gov/pubmed/18365235 PMID: 18365235]
 +
 
 +
 
 +
====Articles====
 +
 
 +
* Tubulin tyrosination navigates the kinesin-1 motor domain to axons. Konishi Y, Setou M. Nat Neurosci. 2009 May;12(5):559-67. Epub 2009 Apr 19. [http://www.ncbi.nlm.nih.gov/pubmed/19377471 PMID: 19377471]
 +
:"Our study identifies a molecular mechanism that discriminates the axonal microtubules from somatodendritic microtubules, as well as a previously unknown linkage between tubulin modification and polarized trafficking in neurons."
 +
 
 +
 
 +
== OMIM ==
 +
 
 +
* [http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=301850 Beta Tubulin]
 +
 
 +
* [http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=191135 Gamma Tubulin]
 +
 
 +
* [http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=602529 Alpha Tubulin]
 +
 
 +
* [http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=157130 Map2]
 +
 
 +
* [http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=157140 Tau]
 +
 
 +
 
 +
== Working area ==
 +
 
 +
Centrioles - cylindrical structures within mitotic spindle pole and act as basal bodies to nucleate the formation of cilia
 +
 
 +
<gallery>
 +
Image:Biotinylated tubulin.jpeg|Tubulin (tem)
 +
Image:Growth cone cytoskeleton.jpeg|Microtubule (fl)
 +
Image:Growth cone cytoskeleton cartoon.jpeg|MT MF Growth cone cytoskeleton cartoon
 +
Image:Myosin_Microtubule_Actin_Collagen.jpg|MT MF Myosin Microtubule Actin Collagen
 +
Image:Centrosome cartoon1.jpg|MT Centrosome cartoon
 +
Image:Mammalian centrosome cartoon.jpg|MT Mammalian centrosome cartoon
 +
Image:Migrating-neuron-cytoarchitecture1.jpg|MT Migrating neuron cytoarchitecture nucleus
 +
Image:Tau tangles cartoon.jpg| MT Tau tangles cartoon
 +
 
 +
</gallery>
 +
 
 +
{{Template:2009ANAT3231}}
 +
 
 +
 
 +
== Movies ==
 +
* '''Tea1p rides on microtubule ends''' [http://jcb.rupress.org/cgi/content/abstract/165/5/697 Feierbach et al.] confirm that Tea1p [http://jcb.rupress.org/cgi/content/full/jcb.200403090/DC1/1 moves to the ends] of fission yeast cells on microtubules. They find that Tea1p does so by attaching to the [http://jcb.rupress.org/cgi/content/full/jcb.200403090/DC1/2 ends] of microtubules, which [http://jcb.rupress.org/cgi/content/full/jcb.200403090/DC1/3 deposit it directly] at the cell surface. Tea1p is probably held on microtubule ends by Tip1p, as in cells lacking Tip1p the Tea1p [http://jcb.rupress.org/cgi/content/full/jcb.200403090/DC1/4 wanders] along microtubules.
 +
 
 +
* '''Microtubule catastrophe under pressure''' [http://jcb.rupress.org/cgi/content/abstract/161/6/1029 Janson et al.] report that force stalls MT growth and induces rapid catastrophes visible as [http://jcb.rupress.org/cgi/content/full/jcb.200301147/DC1/3 single] and [http://jcb.rupress.org/cgi/content/full/jcb.200301147/DC1/4 repeated] events. Detailed measurements suggest the barrier acts simply by slowing tubulin addition, thus giving more time for structural changes leading to catastrophe. This behavior may make microtubules a [http://jcb.rupress.org/cgi/content/full/161/6/1006-a more adaptable positioning device].
 +
 
 +
* '''Dynactin and microtubules search out their organelle targets''' [http://jcb.rupress.org/cgi/content/abstract/158/2/305 Vaughan et al.] visualize the p150''Glued'' subunit of dynactin (a binding partner for cytoplasmic dynein). They find that it associates with [http://jcb.rupress.org/cgi/content/full/jcb.200201029/DC1/1 growing microtubule plus-ends] to form "comet tails." When these tails encounter Golgi-derived membranes, the membranes are seen to initiate [http://jcb.rupress.org/cgi/content/full/jcb.200201029/DC1/6 rapid movement] (see the same phenomenon in [http://jcb.rupress.org/cgi/content/full/jcb.200201029/DC1/7 close-up]). This supports the search-and-capture model in which microtubules probe the cytoplasm for organelles in need of transport. A similar mechanism may underlie microtubule-kinetochore interactions.
 +
 
 +
* '''Tea1p travels to cell ends and keeps polarity factors anchored there''' Tea1p is needed to keep fission yeast growing linearly; in its absence cells become bent and branched. [http://jcb.rupress.org/cgi/content/abstract/157/5/783 Behrens and Nurse] demonstrate that tea1p is [http://jcb.rupress.org/cgi/content/full/jcb.200112027/DC1/1 transported] on the plus ends of microtubules from the vicinity of the nucleus to the cell ends. Tea1p prevents the curling of microtubules around the cell ends, and helps retain polarity factors at the cell ends.
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* '''Cytoplasmic microtubules position the nucleus''' [http://jcb.rupress.org/cgi/content/full/153/2/397/F1/DC1 Cytoplasmic microtubules] in fission yeast run from one end of the cell to another. [http://jcb.rupress.org/cgi/content/abstract/153/2/397 Tran et al.] suggest that these microtubules position the nucleus by attaching to the nucleus and then pushing on the ends of the cell. They first show that the nucleus undergoes microtubule-dependent [http://jcb.rupress.org/cgi/content/full/153/2/397/F6/DC3 deformations], and then that these deformations correlate with growing microtubules [http://jcb.rupress.org/cgi/content/full/153/2/397/F7/DC1 pushing] (see also a [http://jcb.rupress.org/cgi/content/full/153/2/397/F7/DC2 second video]) against the ends of the cell.
 +
 
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* '''Cortex-microtubule interactions position the nucleus and spindle''' [http://jcb.rupress.org/cgi/content/abstract/149/4/863 Adames and Cooper] find that the budding yeast nucleus [http://jcb.rupress.org/cgi/content/full/149/4/863/F1/DC1/1 moves to the mother-bud neck] via capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Subsequent spindle movement [http://jcb.rupress.org/cgi/content/full/149/4/863/F4/DC1/1 into the neck] is mediated by microtubule sliding along the bud cortex, which can sometimes be seen to occur [http://jcb.rupress.org/cgi/content/full/149/4/863/F5/DC1/2 with free microtubules]. In a later paper, [http://jcb.rupress.org/cgi/content/abstract/151/6/1337 Heil-Chapdelaine et al.] found that the cortical protein Num1p provides an essential attachment point for the sliding machinery (see the [http://jcb.rupress.org/cgi/content/full/151/6/1337/DC1/2 color] version, or [http://jcb.rupress.org/cgi/content/full/151/6/1337/DC1/1 grayscale] version with full legend).
 +
 
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* '''Special microtubules for getting into axons''' [http://jcb.rupress.org/cgi/content/abstract/162/6/1045 Nakata and Hirokawa] find that [http://jcb.rupress.org/cgi/content/full/jcb.200302175/DC1/6 preferential transport of cargoes into axons] is directed by a special population of microtubules, which have a high turnover rate and increased binding of the tip-binding protein EB1.
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Revision as of 09:55, 4 April 2011

ANAT3231 Cell Biology online lectures from the 2017 course.

Cytoskeleton - Microtubules

Microtubules

This lecture continues to look at the cell cytoskeleton, covering in detail the microtubule system.

Molecular sizes

Microtubules are the largest filament system of the cytoskeleton and have important functions for intracellular motility of nearly all cytoplasmic structures (organelles, vesicles, and smaller components).

(MH - Note that the role of microtubules in mitosis will not be covered in detail, as this topic is covered elsewhere in lecture series)

The key concepts are: microtubules, intracellular motility, tubulin, microtubule associated proteins, microtubule motors, centrosome, flagella, cilia The lecture slides and textbook alone contain enough information as an introduction to the subject for this level of study. If you are interested in further reading, I have also included below links to more detailed textbooks with further information and images. Please note this additional information is not necessarily examinable, but may be useful if you have not previously studied biology.


Lecture Slides

2010 Microtubule Lecture Slides Lecture11

Objectives

  • Understand the structure and role of microtubules
  • Understand tubulin structure
  • Understand motor molecules of the microtubule network
  • Brief understanding of microtubule associated proteins
  • Brief understanding of drugs that effect microtubules
  • Brief understanding of microtubule role in mitosis

Lecture Overview

  • Microtubules (mt)
  • Structure
  • Formation
  • Function (Will not cover mitosis in detail today, see cell division lecture)
  • Polarity
  • Turnover
  • Associated Proteins (map)
  • Motors
  • Disorders - Alzheimers, cancer therapies


About Microtubules

  • Cell organizing role
  • Cytoskeleton
    • Largest fibre
    • 25 nm diameter
    • cytoplasmic
  • All cells contain
    • Same core structure
    • Same motors
    • Different associated proteins
  • Dynamic
    • Continuous remodelling
  • Movement
    • Intracellular > cellular
    • Cell division


Motility- Intracellular

organelle movement vesicle transport mitosis & meiosis chromosome segregation gene expression transcription factor binding mRNA transport translation protein export transmitter release Motility- Intracellular Axon transported vesicles EM axonal transported vesicles and axonal cytoskeleton in longitudinal section Arrows show rod shaped structures appear as cross bridges between organelles and microtubules Scale bar 100 nm


Basal Bodies of Cilia

Surface of ependymal cell contains basal bodies red rings connected to cilia microtubules longitudinal section Inset: cilia transverse section central MT doublet surrounded by nine MT pairs one of each pair has a hook-like appendage (arrows) Å~100,000 see later motor slides


Endocytic Pathway

Endocytic movement occurs along microtubules can be blocked by drugs Depolymerizing drugs Stabilizing drugs


Fish Pigment Cell Model

Changes in skin coloration in fish Contain large pigment granules (brown) change location in response to neuronal or hormonal stimulus Dispersal and aggregation of pigment granules occur along MTs Movie: GFP-Tubulin Movie: Microtubules and
 Cell Division MT Mitotic Spindle MT Capture of Kinetochores Movie: MT and Yeast


Structure

Long hollow tubes 25 nm diameter Radiate from forming structure Centrosome Spindle pole Basal Body Polarized (+) plus and (-) minus ends Formed from Tubulin 55 kD protein

Microtubule Structure

(A) EM of mt in cross-section ring of 13 distinct subunits Each a separate tubulin molecule an alpha/beta heterodimer (B) EM of a mt assembled in vitro (C) 13 molecules in cross-section (D) side view of a mt tubulin molecules aligned into long parallel rows 13 Protofilaments Each is composed of a series of tubulin molecules, each an a/b heterodimer mt is a polar structure with a different end of tubulin molecule (a or b) facing each end of microtubule

Tubulin Protofilaments

dimers polymerize to form microtubules 13 linear protofilaments head-to-tail arrays of tubulin dimers arranged in parallel assembled around hollow core

Microtubule Polarity

Tubulin subunits in a MT subunits aligned end to end into a protofilament magenta highlight side-by-side protofilament packing forms wall of microtubule slightly staggered so that a-tubulin in one protofilament contacts b-tubulin in neighboring protofilaments

Arrangement of Protofilaments

Singlet typical microtubule tube built from 13 protofilaments Doublet additional set of 10 protofilaments form a second tubule by fusing to the wall of a singlet Triplet Attachment of another 10 protofilaments

Tubulin

  • dimer 55-kd polypeptides
    • α-tubulin (alpha-)
    • β-tubulin (beta-)
  • encoded by related genes
  • third type of tubulin
    • γ-tubulin (gamma-)
  • located at centrosome
  • role in initiating mt assembly

Tubulin Genes

  • human DNA contains about 14 copies per genome of both genes Cleveland et al. (1980)
    • Beta β 6p21.3 - 15 to 20 members
    • Alpha α mainly Chr.12 - 15 to 20 dispersed genes
    • Gamma γ 17q21
  • Also many tubulin pseudogenes

Tubulin Synthesis Regulation

autoregulation in animal cells stability of polysome-bound tubulin mRNAs beta-tubulin RNAs selectively targeted as substrates for destabilization not recognition of specific RNA sequences co-translational recognition of amino-terminal beta-tubulin tetrapeptide after emergence from ribosome Motif could be used in other systems where RNA degradation is coupled to ribosome attachment and translation

Tubulin Homology

FtsZ bacterial GTPase (40,000 Mr) bacterial protein has structural and functional similarities with tubulin ability to polymerize and a role in cell division protein carrying out these ancestral functions in bacteria was modified during evolution to fulfill diverse roles of microtubules in eukaryotes?


Centrosome

slow-growing minus end of MT embedded in centrosome matrix surrounding a pair of centrioles matrix determines number of MTs in a cell By nucleating growth of new MTs Microtubule Organization Movie: Microtubules and Mitochondria Movie: Microtubules and Endoplasmic Reticulum

Centrosome Cycle

Orientation of MTs in Cells (-) Minus ends of MTs generally embedded in a microtubule-organizing center (mtoc) alpha (+) plus ends often located near the plasma membrane beta


Orientation of cellular MT

Neuron- Axon and Dendrites Gamma-Tubulin-mediated assembly of microtubules models for g-tubulin-mediated assembly of microtubules alternative models gamma -TuRC nucleates microtubule assembly either by left- presenting a row of gamma -tubulin subunits right- forming a protofilament, which can directly bind a/b-tubulin subunits

modified from C. Wiese and Y. Zheng, 1999, Curr. Opin. Struc. Biol. 9:250–259.


MT Dynamic Instability

continual and rapid MT turnover half-lives of only several minutes this rapid turnover critical for remodeling of the cytoskeleton during mitosis Tim Mitchison and Marc Kirschner (1984) Movie: GFP mt Movie: GFP mt Movie: GFP mt

MT Treadmilling

Treadmilling dynamic behavior when tubulin bound to GDP continually lost from minus end replaced by the addition of tubulin bound to GTP to plus end of same microtubule GTP hydrolysis also results in dynamic instability individual microtubules alternate between cycles of growth and shrinkage



Microtubule Movement

GTP hydrolysis destabilizes MTs Addition of tubulin adds GTP to end of protofilament grows in linear conformation readily packed into MT wall becoming stabilized Hydrolysis of GTP changes subunits conformation force protofilament a curved shape less able to pack into the MT wall protofilaments with GDP-containing subunits forced linear conformation by lateral bonds within MT wall, mainly in stable cap of GTP-containing subunits


GTP Cap Hydrolysis

GTP hydrolysis destabilizes MTs GDP-containing protofilaments relax curved conformation progressive disruption of MT disassembly of protofilaments free tubulin dimers


MT Stability and cell Polarity

Microtubule Associated Proteins Many different proteins Example - neurons


Microtubule Associated Proteins

Microtubule Associated Protein

  • MAP2
  • Neuron expression
  • a 280-kD protein
  • concentrated in neuronal soma and dendrites
  • Developmentally regulated expression
  • 2q34-q35
  • MAP2 Developmental Expression
    • MAP2B - present throughout brain development
    • MAP2A - appears during end of second week of postnatal life
    • MAP2C - present during early brain development, disappears from the mature brain, except for the retina, olfactory bulb, and cerebellum
  • MAP2A and MAP2B
    • encoded by 9-kb mRNAs
  • MAP2C
    • encoded by a 6-kb mRNA

Tau

  • Protein Mr 45-60 kDa
  • Gene 17q21.1

Neuron Expression Enriched in axons phosphorylated Tau- Alzheimer Disease neuronal cytoskeleton is progressively disrupted replaced by tangles of paired helical filaments (PHFs) PHFs composed mainly of hyperphosphorylated form of Tau Tau- Alzheimer Disease Elevated tau inhibit intracellular transport mainly plus-directed transport (kinesin motors) from center of cell body to neuronal processes organelles are unable to penetrate neurites peroxisomes, mitochondria, and transport vesicles carrying supplies for growth cone Leads to stunted growth increased susceptibility to oxidative stress pathologic aggregation of proteins such as amyloid precursor protein (APP) tau:tubulin ratio is normally low increased levels of tau become detrimental to the cell


Microtubule Motors

Microtubule Motor Proteins

  • Dynein (-) and Kinesin (+) move in opposite directions
  • globular heads of heavy chains bind mts
  • motor domains

Dynein

  • - (minus) end motor
  • 2 or 3 heavy chains (two are shown here)
  • multiple light and intermediate chains

Kinesin

  • + (plus) end motor
  • 2 heavy chains, wound around each other in a coiled-coil structure
  • 2 light chains
  • Movie: Kinesin on Microtubule
  • Movie: Microtubules in vitro

Ciliary and Flagellar Axonemes

  • 9 + 2 MT arrangement
  • dynein arms and radial spokes with attached heads occur at intervals along the longitudinal axis
  • central microtubules, C1 and C2
  • Axonemal Dynein
    • Arrangement of globular domains and short stalks
    • attachment of outer dynein arm to the A tubule of one doublet and cross-bridges to B tubule of an adjacent doublet
    • attachment to A tubule is stable

Presence of ATP successive formation and breakage of cross-bridges to adjacent B tubule leads to movement of one doublet relative to the other Dynein-mediated sliding of axonemal mt Dynein arm attach to A subfiber of one microtubule walk along B subfiber of adjacent doublet toward (-) end (small arrow) moves microtubule in opposite direction (large arrow) cross-links (nexin) broken, sliding can continue


Microtublues In Development

  • nurse cells in insect ovarioles
  • supply oocytes cellular components
  • mRNAs, proteins
  • pass from one cell to another through intercellular bridges traversed by microtubules
  • mRNAs encode axis-determining factors in Drosophila embryos
  • mRNAs are translocated
  • localized within oocyte
  • sites where translation products will function


Microtubule Drugs and Cancer

  • drugs affect microtubule assembly
  • experimental cell biology tool
  • Cancer treatment
  • Colchicine and Colcemid
    • bind tubulin
    • inhibit mt polymerization, blocks mitosis
  • Vincristine and Vinblastine
    • cancer chemotherapy
    • selectively inhibit rapidly dividing cells
  • Taxol
    • stabilizes microtubules rather than inhibiting their assembly
    • also blocks cell division

Taxol - Paclitaxel

  • 1971 from the bark of the Western yew
  • Taxus brevifolia Nut (Taxaceae)
  • Anti-tumor and anti-leukemic activity
  • found in roots, leaves, and stems of this and related members of yew family
  • complex ester
  • an oxetan ring attached to a derivative of taxane
  • tool for investigating MT function
  • clinical trials in a variety of cancers
  • Initial development limited by low abundance in yew trees
  • now novel synthetic methods
  • identification of new sources of taxanes


References

Textbooks

Essential Cell Biology

  • Chapter 16, p518-527

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

Search Online Textbooks

Books

PubMed

  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

2007 Medline search “microtubule” 34899 References, 3523 Reviews

Reviews

  • Structure and function of mammalian cilia. Satir P, Christensen ST. Histochem Cell Biol. 2008 Jun;129(6):687-93. Epub 2008 Mar 26. Review. PMID: 18365235


Articles

  • Tubulin tyrosination navigates the kinesin-1 motor domain to axons. Konishi Y, Setou M. Nat Neurosci. 2009 May;12(5):559-67. Epub 2009 Apr 19. PMID: 19377471
"Our study identifies a molecular mechanism that discriminates the axonal microtubules from somatodendritic microtubules, as well as a previously unknown linkage between tubulin modification and polarized trafficking in neurons."


OMIM


Working area

Centrioles - cylindrical structures within mitotic spindle pole and act as basal bodies to nucleate the formation of cilia

2009 Course Content

Lectures

Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells | Stem Cells | Development | Revision

Laboratories

Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture 1 | Tissue Culture 2 | Microarray Lab visit

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G


Movies

  • Tea1p rides on microtubule ends Feierbach et al. confirm that Tea1p moves to the ends of fission yeast cells on microtubules. They find that Tea1p does so by attaching to the ends of microtubules, which deposit it directly at the cell surface. Tea1p is probably held on microtubule ends by Tip1p, as in cells lacking Tip1p the Tea1p wanders along microtubules.
  • Microtubule catastrophe under pressure Janson et al. report that force stalls MT growth and induces rapid catastrophes visible as single and repeated events. Detailed measurements suggest the barrier acts simply by slowing tubulin addition, thus giving more time for structural changes leading to catastrophe. This behavior may make microtubules a more adaptable positioning device.
  • Dynactin and microtubules search out their organelle targets Vaughan et al. visualize the p150Glued subunit of dynactin (a binding partner for cytoplasmic dynein). They find that it associates with growing microtubule plus-ends to form "comet tails." When these tails encounter Golgi-derived membranes, the membranes are seen to initiate rapid movement (see the same phenomenon in close-up). This supports the search-and-capture model in which microtubules probe the cytoplasm for organelles in need of transport. A similar mechanism may underlie microtubule-kinetochore interactions.
  • Tea1p travels to cell ends and keeps polarity factors anchored there Tea1p is needed to keep fission yeast growing linearly; in its absence cells become bent and branched. Behrens and Nurse demonstrate that tea1p is transported on the plus ends of microtubules from the vicinity of the nucleus to the cell ends. Tea1p prevents the curling of microtubules around the cell ends, and helps retain polarity factors at the cell ends.
  • Cytoplasmic microtubules position the nucleus Cytoplasmic microtubules in fission yeast run from one end of the cell to another. Tran et al. suggest that these microtubules position the nucleus by attaching to the nucleus and then pushing on the ends of the cell. They first show that the nucleus undergoes microtubule-dependent deformations, and then that these deformations correlate with growing microtubules pushing (see also a second video) against the ends of the cell.
  • Cortex-microtubule interactions position the nucleus and spindle Adames and Cooper find that the budding yeast nucleus moves to the mother-bud neck via capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Subsequent spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which can sometimes be seen to occur with free microtubules. In a later paper, Heil-Chapdelaine et al. found that the cortical protein Num1p provides an essential attachment point for the sliding machinery (see the color version, or grayscale version with full legend).


2012 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2012 Revision | Development


Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis



2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G