Cytoskeleton Regulation Proteins


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 Cytoskeleton Regulation Proteins Background

Multicellular organisms are composed of tissues and organs with various architectures that are key to their functions. Tissue architecture, in turn, is largely determined by constituent cell architecture, including global cell shape as well as subcellular positioning of the nucleus and other organelles. The cytoskeleton is responsible for generating and maintaining morphology and organelle organization in cells with a variety of shapes and functions. The cytoskeleton is intracellular filament regulated by various factors, including nucleators, motors, severing proteins, crosslinkers, polymerases and depolymerase. Although diverse, these regulators constitute a conserved basic tool set which work together with a common set of cytoskeletal elements. There are three types of cytoskeleton: microfilaments (actin filaments, often referred as F-actin), microtubules and intermediate filaments. Microfilaments and microtubules are introduced here.

 

Regulation of Microtubule dynamics by motor proteins

One important function of microtubule is intracellular transportation. The polarized microtubules provide an excellent “highway” for directional transport of cargoes. Transportation is carried out by microtubule based motor proteins. There are two types of microtubule associated motors: dynein and kinesin. Dynein is a polypeptide complex that binds to microtubule through the motor domain within the heavy chains. The ATPase activity of the heavy chain allows it to hydrolyze ATP to provide energy for walking along the microtubule filaments. The movement of dynein is minus end directed, meaning towards the microtubule minus end. Besides cytoplasmic localized dynein, there is also axonemal dynein whose activity is essential for cilia or flagella movement. The second type of microtubule associated motor is kinesin. Kinesins comprise a large family of proteins consisting of about 40 kinesins in humans, classified into 14 subfamilies. Like dynein, kinesin also hydrolyzes ATP to power movement along a microtubule. The movement of most kinesins is plus end directed with the exception of kinesin 14 moving towards microtubule minus end. Both kinesin and dynein rely on their light chain to associate with cargoes. Kinesin typically binds cargo through receptors or scaffolding proteins while dynein sometimes requires a class of proteins known as dynactin to mediate the cargo binding. Nonetheless, the light chain of dynein and kinesin can also sometimes directly bind to cargoes.

In addition to their function of transporting cargoes to a specific location within the cells, there is an ever-growing body of evidence suggesting both dynein and kinesin motors have pivotal role in regulating microtubule dynamics. For instance, in the C. elegans one cell stage embryo, dynein is anchored at the cell cortex and binds to microtubules via its heavy chain. The minus-end-directed movement of dynein can generate pulling forces on the microtubule. In the early stage of mitosis the pulling by dynein can rotate the centrosomes and position them along the longitudinal axis of the cell. Thus, dynein is essential for setting up a proper spindle orientation during mitosis. Members of kinesin-13 subfamily present another elegant example of how motor proteins regulate microtubule dynamics. Two members of kinesin-13 subfamily, Klp10A and Klp59C, can recognize microtubule minus and plus ends, respectively and promote depolymerization of microtubule. Depolymerization at the minus end leads to microtubule subunit flux toward the spindle pole (poleward movement), while depolymerization at the plus end, where microtubule captures the kinetochore, will chew up the microtubule track (known as “Pac-Man” model). Both poleward and Pac-Man movements contribute to driving chromatids toward spindle pole during anaphase in mitosis.

 

Actin filaments

Filamentous actin, or F-actin, is another component of the cytoskeleton. It is a polymer of globular actin (G-actin). Monomeric actin is ATP-bond and the ATP will be hydrolyzed when G-actin is incorporated into the actin filament. Like a microtubule, F-actin also has polarity. This is because an actin molecule forms a cleft-like structure to hold ATP, and G-actin monomers are added onto the filaments in such a way that all ADP-holding clefts point to the same direction, designated minus end. There are two types of factors stimulating actin nucleation in the cell. One of them is the arp2/3 complex, a seven- subunit protein complex that is responsible for generating branched actin networks. Arp2/3 binds to the side of an existing actin filament from where it nucleates new filament with a 70° angle on the mother filament. Another type of factor is formin, which nucleates non-branched actin filaments.

Like microtubules, F-actin undergoes cycles of dynamic polymerization and depolymerization that are key to most of its functions. The actin dynamics are regulated by various cellular signaling molecules, among which the Rho GTPases are most notable. Rho GTPases are a family of small G proteins (guanine nucleotide-binding proteins). As implied by the name, Rho GTPases contain a GTP binding domain. GTP-bound Rho GTPase is the active form, and can switch to inactive GDP-bound form. Two classes of proteins, guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAPs), regulate the activity of Rho GTPase. A GEF protein can substitute the GDP of an inactive Rho GTPase with a GTP to convert it into the active form. The GAPs can activate the intrinsic GTPase activity of the G protein, leading to GTP hydrolysis and converting itself into the inactive form. The Rho GTPase family functions as a molecular switches and are the key signaling molecules in regulating actin cytoskeleton by transducing upstream signaling to a broad range of effectors, including WASP, ROCK1, SRA1, etc. Many of these effectors are regulators of actin skeleton.

Three members of Rho family, Rac1, Cdc42, and Rho, have been extensively studied. Each of these G proteins has a distinct role, but they also function in coordination with each other to regulate actin dynamics. For example, active Rac1 can bind to WASP family proteins, such as NWASP and WAVE, who subsequently activate the arp2/3 complex to nucleate branched actin filaments normally found in the lamellipodia of a migrating cell. Active RhoA can lead to the activation of formin whose activity is to nucleate non-branched actin filament. RhoA activity is critical for maintaining stress fibers. It is also the key signaling molecule for assembling actomysin contractile ring during cytokinesis. Cdc42 activity contributes to the formation of filopodia, spike-like membrane protrusions.