Cell bio 8/9

General cellular organization of intermediate filaments

Cytoskeletal filaments ~10 nm in diameter that provide mechanical strength and resist stretching forces in cells

Basic function of intermediate filaments

Provide tensile strength and structural stability to cells and tissues

Common structural motif of IF proteins

Central alpha-helical rod domain that forms coiled-coil dimers

First step of IF assembly

Two monomers form a coiled-coil dimer

Second step of IF assembly

Two dimers align antiparallel to form a tetramer

Higher order IF structure

Tetramers associate laterally to form protofilaments that assemble into ~10 nm filaments

Mechanism regulating IF assembly and disassembly

Phosphorylation causes disassembly while dephosphorylation promotes assembly

Class I intermediate filaments

Acidic keratins found in epithelial cells

Class II intermediate filaments

Basic keratins also found in epithelial cells

Class III intermediate filaments

Vimentin desmin and GFAP found in mesenchymal muscle and glial cells

Class IV intermediate filaments

Neurofilament proteins found in neurons

Class V intermediate filaments

Lamins located in the nuclear lamina

Function of IF associated proteins

Crosslink intermediate filaments link them to other cytoskeletal elements and anchor them to membranes

Effect of stretching on IF tension

Intermediate filaments become stiffer and generate greater resistance as cells stretch

Three main molecular motor families

Myosin kinesin and dynein

Evolutionary polymer track of myosin

Myosin moves along actin filaments

Evolutionary polymer track of kinesin

Kinesin moves along microtubules

Evolutionary polymer track of dynein

Dynein moves along microtubules

Direction of kinesin movement

Typically toward the plus end of microtubules

Direction of dynein movement

Toward the minus end of microtubules

Organization of myosin heavy chain

Two heads neck region with light chains and a long tail that binds cargo

Role of the myosin head

ATP hydrolysis actin binding and force generation

Role of the myosin tail

Binds cargo or allows filament formation

Myosin ATPase cycle first step

ATP binding causes myosin to detach from actin

Myosin ATPase cycle second step

ATP hydrolysis cocks the myosin head into a high energy conformation

Myosin ATPase cycle power stroke

Release of inorganic phosphate triggers force generating movement

Organization of kinesin heavy chain

Two motor heads connected by a neck linker with a tail that binds cargo

Kinesin stepping mechanism

Alternating ATP hydrolysis allows hand over hand movement along microtubules

Diversity of kinesin isoforms

Motor domains are conserved while tail domains determine cargo specificity

Dynein structure

Large multi subunit motor protein with AAA plus ATPase domains forming a ring

AAA motifs in dynein

ATPase domains that drive conformational changes for movement

Overview of the cell cycle

G1 S G2 and M phases that coordinate cell growth DNA replication and division

Function of G1 phase

Cell growth and preparation for DNA replication

Function of S phase

DNA synthesis and chromosome replication

Function of G2 phase

Preparation for mitosis and repair of DNA damage

Function of M phase

Mitotic division of the nucleus followed by cytokinesis

Stages of mitosis

Prophase prometaphase metaphase anaphase telophase

Function of cell cycle checkpoints

Ensure DNA integrity and correct progression before advancing to the next stage

Major cell cycle checkpoints

G1 S checkpoint G2 M checkpoint and spindle assembly checkpoint

Role of cyclins

Regulatory proteins whose levels fluctuate to activate cyclin dependent kinases

Function of cyclin dependent kinases

Phosphorylate target proteins to drive cell cycle transitions

Positive regulation of CDKs

Cyclin binding and activating phosphorylation

Negative regulation of CDKs

Inhibitory phosphorylation and binding of CDK inhibitors

First major family of E3 ubiquitin ligases

SCF complex that recognizes phosphorylated substrates

Second major family of E3 ubiquitin ligases

APC C complex that regulates mitosis progression and exit

Mechanism of SCF activation

Recognizes phosphorylated substrates via adaptor proteins

Mechanism of APC C activation

Activated by regulatory proteins such as Cdc20 or Cdh1 to target cell cycle regulators for degradation

Intermediate filament diameter

Approximately 10 nanometers in thickness which is between actin filaments and microtubules

Reason intermediate filaments are called intermediate

Their diameter is intermediate between microfilaments and microtubules

Mechanical property of intermediate filaments

Highly flexible and able to withstand mechanical stress without breaking

Comparison of IF polarity to actin and microtubules

Intermediate filaments lack polarity unlike actin and microtubules

Effect of lack of polarity in IFs

Intermediate filaments do not support motor protein movement

Major cellular role of intermediate filaments

Maintain cell shape and resist mechanical stress

Where intermediate filaments are often anchored

Desmosomes and hemidesmosomes

Relationship between IFs and epithelial tissue

Keratin IFs provide strength to epithelial layers

Disease associated with keratin mutations

Epidermolysis bullosa simplex

Structural domain shared by IF proteins

Central alpha helical rod domain forming coiled coils

IF monomer structure

Alpha helical rod with variable head and tail domains

IF dimer formation

Two monomers wrap into a coiled coil dimer

Orientation of IF tetramers

Two dimers associate in antiparallel orientation

Importance of antiparallel tetramers

Removes polarity from intermediate filaments

Lateral association of tetramers

Forms protofilaments and protofibrils

Final IF filament composition

Multiple protofilaments packed together into a rope like structure

Strength of IF filaments

Rope like structure provides tensile strength

Role of IF head domains

Regulate filament assembly and interactions

Role of IF tail domains

Provide functional diversity between IF proteins

IF network distribution

Forms a cytoplasmic network extending from nucleus to cell membrane

Location of lamins

Nuclear lamina lining the inner nuclear membrane

Function of nuclear lamins

Provide structural support to the nucleus

Role of lamins during mitosis

Phosphorylation causes lamina disassembly

Effect of lamin mutations

Can cause diseases called laminopathies

Example laminopathy

Hutchinson Gilford progeria syndrome

Example class III IF protein

Vimentin

Cells expressing vimentin

Mesenchymal cells such as fibroblasts

Example IF protein in muscle cells

Desmin

Function of desmin

Links contractile apparatus in muscle cells

Example IF protein in neurons

Neurofilament proteins

Function of neurofilaments

Provide structural support to axons

Relationship between IF abundance and axon diameter

More neurofilaments increase axon diameter

Intermediate filament stability

More stable than actin filaments or microtubules

Energy requirement for IF assembly

Assembly does not require ATP or GTP

Regulation of IF dynamics

Primarily controlled by phosphorylation

Effect of phosphorylation on IFs

Promotes filament disassembly

Effect of dephosphorylation on IFs

Promotes filament assembly

Protein linking IFs to actin

Plectin

Protein linking IFs to membranes

Plakin family proteins

Role of crosslinking proteins

Organize IF networks and connect cytoskeletal systems

Definition of molecular motor protein

Protein that converts ATP energy into mechanical movement

Three main cytoskeletal tracks

Actin filaments microtubules and intermediate filaments

Motor proteins that move on actin

Myosin family

Motor proteins that move on microtubules

Kinesin and dynein

Energy source for molecular motors

ATP hydrolysis

General function of motor proteins

Transport cargo within cells and generate force

Myosin evolutionary origin

Actin based motor family conserved across eukaryotes

Kinesin evolutionary origin

Microtubule based motor family conserved across eukaryotes

Dynein evolutionary origin

Large microtubule motor unique to eukaryotes

Direction of kinesin movement

Toward microtubule plus end

Direction of dynein movement

Toward microtubule minus end

Example cargo transported by kinesin

Vesicles organelles and protein complexes

Example cargo transported by dynein

Endosomes lysosomes and chromosomes