Intermediate Filaments

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Properties of Intermediate Filaments

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present in only animals such as vertebrates, nematodes and arthopods
-localized to some cells
-not present in oligodendrocytes
-are not involved in cell movement and motility
-impart physical strength to cells and are associated with cell membranes
Divergent in evolution
-do not rupture in response to great stress (relatively to other filaments)

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Assembly of IFs

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o Elongated molecules with extended central alpha helical domain forming parallel coiled coil with another monomer
o Pair of parallel dimers then associates in antiparallel fashion to form staggered tetramer
o Ends of tetramer are the same and lacks structural polarity like MTs and AFs
o IF has 8 parallel protofilaments made of 32 helical coils and has strong hydrophobic interactions

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Structure of IFs

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Subunits are alpha helical rods that assemble into ropelike filaments and do not involve ATP or GTP hydrolysis
• \\Has a long central alpha helical region flanked by head (amino terminal) and tail (carboxyl terminal) domain of a more flexible and more variable structure
• The non-helical head and tail domain are made of three distinguishable regions. These include E1 (head) and E2(tail), the extreme subdomains are highly charged V1(head) and V2 (tail) are variable domains containing loose repeating sequence motifs which are similar in proteins expressed in similar tissue sites and H1(head) and H2 (tail) are hypervariable stretches that contain phosphorylation target sites.

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assembly group 1

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Keratins assemble from heterodimeric tetramers by lateral and nearly simultaneous assembly into heterogenous full-width filaments

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assembly group 2

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Vimentin type assembly starts from anti-parallel, half-staggered double dimers (tetramers) to form full width, unit-length filaments (ULF)

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assembly 3

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Lamin dimers associate first into head-and-tail filaments that later laterally associate.

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Keratin

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Most diverse IF family and found in skin, hair,nails,claws and scales

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formation of keratin

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Each keratin filament is made of equal mix of Type I (acidid) and Type II (basic/neutral) forming a hetrodimer
o Two hetrodimers join together to form a tetramer
o Cross-linked keratin networks are held together by disulphide bonds surviving after death of cell

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function of keratin

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Keratins are found throughout the entire length of the keratinocytes and between cells by attachment of desosomes cell-cell junction and between basal cells and basal lamina by attachment of hemidesosomes.

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ALS

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Amyotrophic lateral sclerosis (ALS) is associated with accumulation and abnormal assembly of neurofilaments in motor neuron cell bodies and in axon
Axon degeneration leads to muscle weakness and atrophy

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function of neurofilaments

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the radial growth of axonal processes requires the interaction of neurofilaments to exhibit correct proportions of light, medium and heavy subunits
o large C terminal C-terminal tail domains of NF-H and NF-M subunits are hyper-phosphorylated and project away from the filament core, thereby determining inter-filament spacing and axonal calibre.
o The many neurofilaments interact with microtubules and with subcortical actin filaments through cytoskeletal linker proteins, such as plectin and BPAG1.

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function of vimentin

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After chemokine-induced chemotaxis at sites of active inflammation, vimentin intermediate filaments are rapidly move to the perinuclear region at the cell uropod by the site-specific phosphorylation of vimentin
o subunits correlates with a softening of the viscoelastic properties of the cytoplasm, presumably to allow the pliability needed during extravasation and also apply to the ability of epithelial cells to migrate into a wound site after injury.

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role of IF in cellular signaling

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Cytoplasmic intermediate filaments can bind and modulate the activity of signalling proteins, thereby influencing the flow of extracellular signals to relevant terminal effectors inside the cell.
o interactions of intermediate filaments with cell surface receptors, such as Fas, can modulate their density and function
o Regulated interactions between intermediate filaments and an adaptor protein, such as TRADD, near the cell surface limits the availability of this adaptor to a ligand-bound receptor that is prepared to transmit a signal to the cell.
o Regulation of either type of interaction can be mediated by dynamic post-translation modifications, association with other proteins or local differences in the composition of intermediate-filament subunits.

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loss of vimentin

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significantly reduced rate of dermal fibroblast migration resulting in retarded myofibroblast formation and wound contraction

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loss of keratin 6

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significantly reduced rate of dermal fibroblast migration resulting in retarded myofibroblast formation and wound contraction

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loss of keratin 8 and 18

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Loss of cell polarity (in mice defects in IF proteins lead to mistargeting of apical and basolateral membrane proteins in gut epithelial lining --> Leads to bowel disease).
o Affecting susceptibility of hair follicle cells to apoptosis (by sequestering the proteins required in apoptotic signaling pathway).

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loss of lamin a and c

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Various disorders (including some types of muscular dystrophy)
o Alterations in IF gene expression are known to occur in some cancers.

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upregulation of keratin 17

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Keratin 17 expression is upregulated in basal cell carcinoma and in a type of breast cance

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initial tissue replacement phase

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Alterations in IF gene expression patterns facilitating movement of cells proximal to the wound edge into the site of injury
-Transition of activated cells distal to the wound edge to a hyperproliferative state

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remodeling phase

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o Expression pattern of IF genes reverts back to normal state
o Cell differentiation begins to reestablish tissue architecture

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what do basal cells express

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, basal layer cells are mitotically active and mainly express keratins 5 and 14.

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what do suprabasal cells express

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Suprabasal layer cells that are composed of keratinocytes at various points of differentiation, predominantly express keratins 1 and 10.

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oligodendrocytes

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: a special type of Glial cells of nervous system which do not contain intermediate filaments

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hemidesosomes

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are structures on the inner basal surface of keratinocytes in the epidermis of skin and attach one cell to the extracellular matrix. They are asymmetrical and are found in epithelial cells connecting the basal face to other cells.

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plectin

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a giant protein found in nearly all mammalian cells which acts as a link between the three main components of the cytoskeleton: actin microfilaments, microtubules and intermediate filaments.

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desmin

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intermediate filament found near the Z line in sarcomeres.

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vimentin

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IF that plays a role in supporting and anchoring the position of the organelles in the cytosol specially mitochondria and ER

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structure of molecular motors

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he head domains bind to a cytoskeletal fiber (AFs or MTs) and the tail domain attaches to a cargo. ATP hydrolysis provides energy for “walking”.

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function of motors

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motors

vesicles, organelles (mitochondria, lysosomes, chloroplasts), chromosomes, other cytoskeletal filaments transport

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describe mechanochemical cycle

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Motor proteins move unidirectionally along their cytoskeletal track in a stepwise manner from one binding site to the next
As they move along, they undergo a series of conformational changes (a mechanical cycle)
Involves ATP binding and hydrolysis (a chemical cycle)
Steps of mechanical cycle are coupled to chemical cycle
Binding & hydrolysis of one ATP molecule moves motor a few nm along track; Cycles repeated many times

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Myosin move toward _____ end of Actin filament except Myosin ___

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plus, VI

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myosin structure

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All share characteristic motor (head) domain, which has a site that binds actin filament and one that binds and hydrolyzes ATP to drive the motor
While head domains of myosins are similar, tail domains are highly divergent
Myosins also contain variety of low molecular weight (light) chains

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types of contractile bundles involved by actin-myosin 2

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Circumferential belt
Encircles the inner surface of the cell
Stress fibers
Seen along the ventral surface of cultured cells on artificial glass (or plastic)
Contractile rings
Transient structures responsible for cytokinesis

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define yeast vacoule inheritance and yeast vacoule

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The yeast vacuole inheritance is a model to understand the mechanism of organelle transport inside eukaryotic cells (during cell division).
Yeast vacuole is an endocytic organelle (equivalent to lysosome) that plays important role in ion and water homeostasis, storage of metabolites, and protein turnover.

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structure of myosin V Myo2 complex

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The motor domain contains an actin-binding region and an ATPase. The IQ motifs, which
form the neck of each molecule, bind calmodulin and other regulatory light chains. The
coiled-coil region is required for dimer formation, and the globular tail domain is the
cargo-binding region

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components of myo2 complex

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Myo2, the vacuole-specific adaptor/receptor
Vac17, and a myristoylated and palmitoylated vacuolemembrane
protein Vac8

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explain how myo2 complex mediates organelle transport

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Initiation:Vac17 synthesis is coordinated with the cell cycle. Vac8 and Myo2 are present throughout the cell cycle and initiation probably occurs through the formation of the Myo2–Vac17–Vac8 complex
Movement:The Myo2–Vac17–Vac8 transport complex moves the vacuole to the bud,
Termination: the complex is disrupted by the regulated turnover of Vac17. This regulated turnover deposits the vacuole in its proper location and might also release Myo2 to move other cargoes.

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kinesins

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-motor protein that moves along microtubules.
-Kinesins move vesicles/organelles from cell body to synaptic knobs
First isolated in 1985 from squid giant axons
A tetramer made of two identical heavy chains and two identical light chains
A pair of globular heads generates force by hydrolyzing ATP and binding MT

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Explain the in vitro mobility assay of Kinesin Movement

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kinesin-coated beads move towards "+" end (axon tip); therefore, kinesin responsible for anterograde movement
All MTs of axon are oriented with"-" ends facing cell body and "+" ends facing synaptic knobs
- kinesin moves towards plus end like myosin

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explain how swinging movement of neck results by kinesins

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Kinesin possesses two motor domains that work by “hand-over-hand” mechanism; one always firmly attached to MT
Two heads of kinesin behave in coordinated manner, so that they are always present at different stages in their chemical & mechanical cycles at a given time
When one head binds to MT, the interaction induces a conformational change in adjacent neck region of motor protein; it swings the other head forward toward binding site on next dimer
Force generated by head catalytic activity leads to swinging movement of neck

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dyenins

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family of minus end directed microtubule motors

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types of dyneins

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-cytoplasmic dyenins
-axonemal dyenins

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structure of dyenins

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Two identical heavy chains and a variety of intermediate & light chains
Each dynein heavy chain forms large globular head (~10X larger than a kinesin head) that generates force;

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role of cytoplasmic dyenins

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Force generating agent for chromosome movement in mitosis
"-” end directed MT motor for Golgi complex movement of vesicles/organelles through cytoplasm
In nerve cells, cytoplasmic dynein involved in axonal retrograde organelle movement (toward cell body)

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does dynein act alone?

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Cytoplasmic dynein does not interact directly with membrane-bounded cargo, but requires intervening multisubunit complex, dynactin, that may regulate dynein activity and help bind it to MT
-dynactin complex includes short actin filament that forms from Arp1
-Arp 1 may mediate attachment of the complex to membrane enclosed organelles through network of spectrin and ankyrin

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problems with current dyenin-dynactin model

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Present model may be overly simplistic: kinesin & cytoplasmic dynein move similar materials in opposite directions over the same railway network

Individual organelles may bind kinesin and dynein simultaneously although only one is active at given time; myosin may also be present on some of these organelles

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explain the neural intermediate filament transport model

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A model for neural intermediate filament transport. Neural IF proteins in the form of nonfilamentous particles, squiggles and longer IF move rapidly along microtubles (blue) in association with kinesin (yellow) and cytoplasmic dynein and dynactin (orange). These motors are responsible for the timely delivery of neural IF particles and squiggles, the precursors to the long IF, to all regions of the neuron including the growth cone (on the right side of cell). Long neural IFs also move along neurites, albeit more slowly than the precursors. Their
slower motility might be due to associations with IF-associated proteins such as plectin and bullous pemphigoid antigen (BPAG) (red). In
addition, a population of neural IF proteins may move along actin structures (purple) in association with myosin Va (pink) in the cortical and peripheral domains of the growth cone.
-Because there is a
continuous tug-of-war between actin- and MT-based motors, myosin Va could act as a brake for NF
transport that antagonizes the overal