BIOLOGY 2B03 Chapter Notes - Chapter 7: Actomyosin Ring, Phalloidin, Ovarian Cancer

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BIOLOGY 2B03 - Module 7 Lecture I
Cytoskeleton is Highly Organized
Cytoskeleton provides cell shape and structure
Specialized cell structures is necessary in differentiated cells
Cell shapes depend on the functions of different filaments
Cytoskeleton is Dynamic
Dynamic to provide movement
ex) migration of cells and cell division
animation) ovarian cancer cell migration on glass; cell is expressing Actin-GFP
Breast cancer cell undergoing cell division; expressing Tubulin:GFP
Cytoskeleton is a System of Filaments and Tubules
Three classes of filaments in eukaryotic cells
Defined by diameter and the type of subunit used as building blocks for the filament
Actin filaments are labeled with fluorescently-tagged phalloidin molecule
Phalloidin molecule: toxin derived from a mushroom (death cap)
Bind to actin monomer with high affinity and specificity
Stabilized the filament when bound
Actin can be also labeled with antibody or with a protein fusion (Actin:GFP)
Microtubules are labeled with antibodies specific to the subunits or protein fusion
(Tubulin:GFP)
Intermediate Filaments (IFs): can be labeled as antibody specific to a monomeric subunit of
the filament of GTF-fusion
They all form overlapping
Cytoplasmic Fibres are Polymers
Each filament is constructed from smaller protein subunits to form a long polymer
Actin filament: thinnest filament and are composed of monomeric actin subunits
Microtubules: thickest filaments and are made up of dimeric subunits of alpha- and beta-
tubulin
There are many different IFs; each is assembled from a different protein or set of proteins
Filament Distribution
Three different ways of the filaments distributions in each cell type
Epithelial cell: all three types of filaments are seen in unique locations
Actin: microvilli shape at the apical cell surface
IFs: provide structural support by spanning the cell
Made with lamin proteins form the nuclear lamina: provides structure and
shape to the nucleus
Microtubules: networks for intracellular
transport
Motor Proteins Track on Filament Highways
There are filament-specific motor proteins
Track along actin filaments and microtubules
No motor proteins identified for IFs
Myosin Protein: move along actin filaments
Kinesin and Dynein: track along microtubules
The head domains bind to a cytoskeletal fiber (AFs or
MTs) and the tail domain attaches to a cargo
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ATP hydrolysis → provides energy
Actin-based Structures and Movement
Highest density of actin is at the cell periphery
Actin filaments underlying the cell membrane determine the shape and movement of the cell
surface
Typical functions: the establishment of microvilli, the formation of contractile bundles that
form sarcomeres, formation of filopodia and lamellipodia
Sarcomeres: power muscle cell contraction
Filopodia and lamellipodia: needed for cell migration and the contractile ring that
directs cytokinesis
Actin Filaments are Assembled from Monomers
Actin filament (F-actin, EM) are two-stranded helical polymers
Total diameter 5-9nm
Each polymer is built from actin monomer called G-actin
Actin Filaments are Polar
Two ends look and behave differently from one another
Actin filaments have been decorated with a portion of the actin-binding
protein, myosin
Myosin: its head protein binds in just one orientation on the actin filament
Proteins point away from the actin filament
Able to define plus and minus end based on the rate of actin
polymerization
The plus end: grows more quickly through addition of more actin subunits
and has barbed appearance
The minus end: grows more slowly and may shrink, has a pointy appearance
G-actin Monomer
G-Actin: a single actin monomer
Can be divided into four structural domains with a large cleft between
domains 2 and 4
The cleft forms an ATP-nucleotide binding site
Each of the is polar → the microfilaments built up from these are also
polar
The ATP-binding pocket is pointed to the minus-end
ATP-binding pocket of each monomer is not exposed within a
filament
Except for a pair of monomers right at the minus-end
F-actin: Actin Polymerization and Depolymerization
Filamentous Actin: is created through the polymerization of actin monomers
Note: actin filament is not a static but dynamic structure
Constantly engaged in polymerization and depolymerization
Tendency to be more growth at the plus-end and more shrinkage at the minus-end
ATP-binding regulates the growth and disassembly of the actin filaments
Enough [actin-ATP] in
cytosol → able to join
the plus end
Actin has an intrinsic ATPase activity
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Document Summary

Specialized cell structures is necessary in differentiated cells. Cell shapes depend on the functions of different filaments. Ex) migration of cells and cell division. Animation) ovarian cancer cell migration on glass; cell is expressing actin-gfp. Breast cancer cell undergoing cell division; expressing tubulin:gfp. Cytoskeleton is a system of filaments and tubules. Three classes of filaments in eukaryotic cells. Defined by diameter and the type of subunit used as building blocks for the filament. Actin filaments are labeled with fluorescently-tagged phalloidin molecule. Phalloidin molecule: toxin derived from a mushroom (death cap) Bind to actin monomer with high affinity and specificity. Actin can be also labeled with antibody or with a protein fusion (actin:gfp) Microtubules are labeled with antibodies specific to the subunits or protein fusion (tubulin:gfp) Intermediate filaments (ifs): can be labeled as antibody specific to a monomeric subunit of the filament of gtf-fusion. Each filament is constructed from smaller protein subunits to form a long polymer.

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