3. The Cytoskeleton

Microtubule Polarity

+ and - ends

*Gain an understanding of the basic rules governing the assembly of cytoskeletal systems* 2 general systems of nucleation mechanisms:

1. Arp2/3 complex -- generates specific type of actin filament organization. Requires an existing filament to begin. Arp2/3 binds to the side of an existing filament. Begins to nucleate another filament that grows as a branch at approximately 70 degrees Generates *branched* types of actin filaments 2. Foramins / Spire -- do not need an existing filament Start by grabbing two actin monomers Organize them and bind them to each other Nucleate the filament Typically generate *linear bundles* of filaments

Microtubules are Composed of ____________________?

13 (+) Protofilaments Protofilaments -- 'stacks' of tubulin dimers The plus end is where the dynamic instability occurs The tip of a growing microtubule usually has a GTP loaded in the end--GDP along the shaft

Immotile cilia syndrome

A body-wide defect in axonemal structure that result in obstructive lung disease and sterile males. no mucalarry escalator system

Kartagener's syndrome

A combination of Situs inversus (reversal of normal body asymmetry) and Immotile cilia syndrome Heart on wrong side, etc.

The Basal Body

A microtubule organizing center for cilia and flagella The basal body is at the negative end of the microtubule in cilia

Cytoskeleton

A system of protein polymers that provide for the architecture, shape, and motility of cells and for the directed movement of organelles and molecules within the cell

Neurodegenerative

Abnormalities/mutations in tau, dynein, kinesin, disorders spastin Not properly cut

Microvilli

Actin based epithelial projections Small intestine: increase surface area for absorption during digestion Ear cells: detect sound waves using stereocilia

3 Actin isoforms:

Alpha -- expressed almost exclusively by cells of *muscle* lineage Beta -- *ubiquitous* similar to Gamma Gamma -- *ubiquitous*

Phalloidin

Attacks actin cytoskeleton. Binds to and stabilizes actin filaments

Microtubules Associated Motor Proteins

Both have head domains--contain microtubule binding domains. Both motors are ATPases. Associated polypeptide chains can have control over how fast motor is moving, OR what the motor is going to attach to. KINESIN: (+ end directed) -- move cargo toward the plus end. DYNEIN: (- end directed) -- major retrograde motor protein. *Dynein carries things toward the center of the cell*.

Centrosome Structure

Cell has 2 small microtubules that are perpendicular at the center Gamma tubulin is found almost exclusively in centrosome

Microtubules in Cell Division

Centrosome Microtubules, DNA Microtubules Make spindle fibers Functions: segregation of Chromosones, reorganization of cytoplasm Required components: motor proteins, MT dynamics

Which microtubule based structures are used for movement?

Cilia and Sperm Tails (flagella)

Microtubules: Composition

Composition Polymers of *tubulin dimers* made up alpha-tubulin and beta-tubulin Tubulin is a *GTPase (hydrolyzes GTP)* Hollow with an outside diameter of 24 nm Accessory proteins called microtubule-associated proteins (MAPs) stabilize and space the polymers and regulate interactions between cytoskeletal elements

*Gain and understanding of the differences between cytoskeletal systems* Microfilaments (aka Actin filaments): Composition *Differences between actin and microtubules*

Composition: Non-hollow polymers of the globular protein *actin* Actin is an *ATPase* (as opposed to GTPase) Helical in structure (two chains wrapping around each other) Roughly 7 nm in diameter

Myosins: F-actin Associated Force Generating Mechanoenzymes with Roles in Contractility and Intracellular Transport

Contractility of muscles

Hereditary spherocytosis

Deforms red blood cells to fragile spherocytes because of weakened binding affinity of spectrin to Band 4.1

Hereditary elliptocytosis

Deforms red cells to fragile elliptocytes because of incomplete formation of spectrin

Diseases Associated with Malfunction ofIntermediate Filament Based Cellular Structures

Epidermolysis bullosa Progeria 'fast aging disease'

Epidermolysis bullosa

Epidermolysis bullosa simplex mutation in keratin genes expressed in basal cell layer of epidermis, result in a skin that is very sensitive to mechanical injury.

Cytokinesis

F-actin amd myosin II at the Contractile Ring During Cell Division

Erythrocyte (red blood cell) cytoskeleton

F-actin serves as scaffold for spectrin web The structure of the spectrin web is determined by the ability of the spectrums to form nodes within the web Actin is present in these nodes

Microtubules: Function

Functions *Make up the mitotic spindle* Provide railways for organelle transport in most interphase cells Key determinant of cell shape Very important in neurons for cell shape and axonal transport Provide the backbone for cilia and flagella

Microfilaments (aka Actin filaments): Functions

Functions Concentrated in cell cortex for various functions Cleavage furrow for pinching off cells in final stages of mitosis Cell motility Short-range organelle transport Contractility (in both muscle and non-muscle)

*Intermediate Filaments*: Functions

Functions Space-filling elements Give cells tensile strength Specialized functions depending on cell type Important at cell junctions

Microtubules Associated Proteins (structural non-motor proteins) Don't confuse structural with motor

Functions: organize MTs, regulate MTs stability, Regulate MT dynamics.

Diseases Associated with Malfunction of Actin Based Cellular Structures

Hereditary spherocytosis Hereditary elliptocytosis Breast cancer Familial hypertrophic cardiomyopathy

Diseases Associated with Malfunction of Microtubule Based Cellular Structures

Immotile cilia syndrome Kartagener's syndrome Cancer Lissencephaly Charcot-Marie-Tooth Neurodegenerative

*Intermediate Filaments*: Important point

Important point: Much less conserved across cell types than microtubules or microfilaments Different cell types have different kinds of intermediate filaments (can be used as cell-specific "markers")

The Axoneme

In flagellum 9+2 microtubule structure of cilia and flagella Have 9 "doublets" (2 microtubules fused together) Dynein drives axonemal motility

Components of the cytoskeleton

Microtubules, microfilaments (aka Actin filaments), intermediate filaments, and a host of accessory and regulatory proteins

Charcot-Marie-Tooth

Mutation in *specific kinesin* has been reporteddisease type 2A in one case (other families have mutations in mitofusion2, a protein of mitochondria).

Familial hypertrophic cardiomyopathy

Mutations in a specific cardiac actin.

Lissencephaly

Mutations in the microtubule proteins LIS1 and doublecortin. The brain does not develop typical gyri and sulki -- *"smooth brain syndrome"*

Mutations/alterations in myosin underlie diseases.

Myosin VI mutations cause deafness. Myosin VII mutations associated with deafness, neurological disorder, and blindness (Usher syndrome Type 1)

__________________ exploit the neuron's microtubule based transport system to reach cell bodies

Neurotopic viruses Viruses can use microtubules to attack the cells. Can be incorporated into an endosome, or incorporated on the motor proteins.

Rescue

Period where microtubule begins polymerizing (growing) again

Actin Filament Dynamics

Polymerization is ATP hydrolysis dependent barbed end is dynamic Must be ATP loaded pointed end has less going on

Progeria

Progeria 'fast aging disease' associated with a mutation in nuclear lamin protein

Microtubules: Properties

Properties Highly dynamic if not stabilized; can undergo rapid bouts of assembly and disassembly Act as substrate for microtubule-based motor proteins to transport cargo (e.g., organelles)

Microfilaments (aka Actin filaments): Properties

Properties: *Highly dynamic* if not stabilized; undergo rapid bouts of assembly and disassembly Huge array of configurations regulated by accessory proteins (examples: bundles and meshworks) Act as substrate for members of the myosin family of motors to move along and carry cargo Unlike microtubules actin filaments do not have specific organizing centers like the centrosome and can be nucleated almost anywhere in the cell Used to move things locally (short distances)

Cytoskeleton Components

Protein Polymers Actin -> actin filaments Tubulin -> microtubules Intermediate filaments

Catastrophe

Rapid Shrinking of a microtubule

Functions of Actin Associated Proteins

Regulation of actin--proteins will bind to sequester the actin molecules Or load with ATP to promote regulation Can cap the filaments Severe Can organize the filaments

Gamma tubulin

Required for the initiation of nucleation. Required for the assembly of alpha/beta dimers into micro tubular like structure

Breast cancer

Some forms the actin associated protein Tensin, which links integrin receptors to the actin cytoskeleton, is disrupted promoting metastatic migration of cancerous cells

*Intermediate Filaments*: Structure

Structure *Non-polarized* Roughly 10nm in diameter Comparatively NON-dynamic (much more stable)

*Microtubules: Structure*

Structure *Polarized polymer* with a plus-end and a minus-end *"Plus end is where the action happens--growing and shrinking"* Plus end is dynamic (lengthens and shortens) Motor proteins read the polarity and move to one end or the other of the microtubule

Microfilaments (aka Actin filaments): Structure

Structure Polarized filament with barbed end (+) and pointed end (-) Barbed end favored for assembly over pointed end Myosin motors read the polarity and move toward one end or the other (most myosins move toward plus end) Barbed end is more dynamic

Cancer

Target for therapeutics, e.g. disruption of MT dynamics to block cell division (e.g., Taxol)

Polymerization

The elongation of a cytoskeletal polymer following the initial nucleation

Nucleation

The genesis of a cytoskeletal polymer A gamma tubulin mediated event Often occurs at the centrosome

What comprises the cytoskeleton?

The skeleton and muscles of cells

Cytoskeleton and Cell Attachment to the Extracellular Matrix (ECM)

There is an indirect physical link made by multiple proteins made directly to the actin filaments inside the cell Forces can be communicated from inside to outside or from outside to inside

Where are actin filaments nucleated?

Unlike microtubules, actin filaments do not have specific organizing centers like the centrosome and can be nucleated almost anywhere in the cell

Microtubules: The Highways of the Cell

What a motor protein looks like

Actin Filament Nucleation

anytime, anywhere Start out with soluble actin, and start the filament

Most myosins move toward the _______ end of filaments

barbed

In microtubules, nucleation occurs at the _______________?

centrosome

2 types of myosin molecule

monomeric -- one molecule dimerics -- 2 In all cases there are the long chains Essential Chains--required for basic function Regulatory Chains--subjected to phosphorylation --> when phosphorylated, allow motor to be active