Actin and Myosin

Describe the function of unconventional myosin. What are some physiological roles of these myosin?

1. *Move cargos* (vesicles, organelles, protein complexes) on actin filaments. a. Myosin V moves melanosomes into processes of melanocytes for the delivery of melanin to basal keratinocytes of the skin. Defects in myosin V cause *albinism*. b. Several different myosins are involved in positioning sound transduction components in hair cells of the inner ear. *Defects in these myosins cause deafness*.

Describe conventional (type II) myosin. What is their role and the structure of the filaments.

1. Move actin and are responsible for muscle and stress fiber contractions. 2. Bind two light chains and have tails that can dimerize. 3. Type II myosin tails can further oligomerize into filaments. 4. Polymerized type II myosin = "thick filaments". Have a bipolar organization that orients the head domains toward the ends of the thick filament (Fig 7).

Describe smooth muscle myofilaments and the properties of contraction in smooth muscles.

1. Smooth muscle myofilaments are used in smooth muscle cells, myofibroblasts and myoepithelial cells. 2. These cell types express smooth muscle actin gene products and have myofilament systems organized around dense bodies. Myofilaments radiate out from dense bodies, connecting to other dense bodies and to anchor sites on the plasma membrane. *Contractions of smooth muscle myofilaments thus cause contraction in all directions.* 3. Because smooth muscle myofilaments have *fewer myosins per thick filament* than skeletal or cardiac myofilaments, they *generate less force*; however, *the thin filaments in these cells are much longer* than in cardiac/skeletal muscle allowing smooth muscle thick filaments to make *longer contractions*.

Describe properties of stress fibers.

1. Stress fibers are present in most cells where they mediate the *contractions that alter cell shape and drive cell motility*. 2. Stress fibers use different actin & myosin gene products than those used by muscle cells. 3. The thick filaments of stress fibers have fewer myosins than thick filaments of skeletal/cardiac muscle sarcomeres, but like smooth muscle myofilaments, stress fibers have long actin filaments and are capable of large contractions. 4. Stress fibers do not involve dense bodies, but do *make contacts with the plasma membrane through focal adhesions and adherens junctions*.

What is the myosin work cycle?

1. The start of the cycle begins with nucleotide free myosin weakly bound to actin. 2. *ATP binding releases myosin from actin.* 3. *ATP binding and hydrolysis* drives a conformational shift between the head and neck to *an extended (cocked) state*. Both ADP and organic phosphate remain bound. 4. Adoption of the *cocked state reactivates actin binding*. 5. *Binding of myosin* to actin triggers *release of phosphate*. 6. *Release of phosphate strengthens actin binding and releases myosin from the cocked state.* The conformational change back to the starting state drives the power stroke of the work cycle. 7. *Re-adoption* of the starting conformation *triggers ADP release* and weakens actin binding. This completes the cycle.

Explain Actin assembly/disassembly i.e. explain the thread milling effect.

Actin is an ATPase whose activity is greatly increased when the barbed end surface is occupied by another actin molecule as occurs within a filament. As a consequence, most actin molecules in filaments are in an ADP state with the exception of the actin at or near the barbed end.Actin monomers that are free in the cytosol preferentially bind to the barbed (ATP- bound) end. This ATP dependence helps make the barbed end the fast growing end. G-actin levels in the cytosol are near the critical concentration for polymerization at the barbed end, but are below the critical concentration for polymerization at the pointed end. Consequently, actin has a propensity to dissociate from the pointed end. *Tread-milling is a process in which the overall length of an actin filament remains constant, but the filament moves like a conveyor belt through coincident assembly at the barbed end and disassembly at the pointed end.* This process provides a means of locomotion for certain internal components.

What are actinomyosin filaments?

Actinomyosin filaments are composed of both F-actin and polymerized myosin. Actinomyosin filaments mediate contractions. In muscle, actinomyosin filaments are composed of both thick and thin filaments and are termed myofilaments.

What are actin filaments?

Also termed *F-actin or microfilaments*. Actin filaments are polymers of actin monomers (G-actin). Actin filaments form the core of thin filaments in muscle cells.

What does Thymosin-β4 do?

Binds to *both the pointed and barbed ends* of actin monomers (G-actin). This binding prevents association of G-actin with either the pointed or barbed ends of actin filaments and thereby effectively *reduces the pool of G-actin* that is available to incorporate into filaments.In this way, thymosin *allows cells to maintain a total G-actin level in the cytosol that is much greater than the critical concentration* (the concentration at which G-actin will polymerize into filaments).

What is the corticol skeleton?

Cortical skeleton refers to cytoskeleton at the cell periphery. The membrane skeleton is the part of the cortical skeleton that is closest to the membrane. The cortical skeleton also includes an extensive intermediate filament network below the membrane skeleton. *Support role of F-actin.*

What is cytokinesis? What is the role of actin in this process?

Cytokinesis is the final stage of cell division, when the two daughter cells separate. *Actinomyosin filaments* form a ring structure at the point where fission will occur. Contractions in this ring structure pinch the plasma membrane prior to scission.

What are filopodia?

Filopodia - antenna-like extensions that detect and respond to chemotactic factors. *Made of actin filaments*

What are fibrin?

Fimbrin is a non-extensible actin cross-linking protein with two actin binding sites. It binds *parallel* actin filaments into *tight* actin bundles. Makes the smallest actin bundles.

Describe the MLCK/MLCP system. In what "structures" is this system important?

In the response to calcium or other signals, MLCK (myosin light chain kinase) phosphorylates the light chains of myosin. This phosphorylation promotes the ability of type II myosin to form thick filaments and binds to actin. Once activated, these thick filaments will continue to work until deactivated by MLCP (myosin light chain phosphatase). This system is important for the contraction of smooth muscle and stress fibers.

What is Spectrin? What does a mutation in spectrin cause?

Is an extensible tetramer consisting of a head-to-head dimer of two α/β-heterodimers (Fig 5). Has two actin-binding sites, one at the N-terminus of each β-subunit, thereby positioning the actin-binding sites at either end of the tetramer. *Makes the most loosely connected actin fibers*. Loss-of-function mutations in erythrocyte spectrins are a common cause of *hemolytic anemia*.

What is the structure of Actin Monomers (G-actin)?

Is asymmetric with a minus (pointed) end and a plus (barbed end). Has 4 domains and an ATP cleft. Note: *ATP hydrolysis is slow in monomer, but rapid in filament.*

What is the membrane skeleton? What happens when the membrane skeleton is not stable?

Is composed of actin filaments cross-linked by elastic proteins, principally α-actinin and spectrin. Underlies the plasma membrane and is critical for plasma membrane stability and function. Example 1: Defects in the membrane skeleton of erythrocytes destabilize the red cell plasma membrane. This destabilization causes premature destruction of red cells, resulting in *hemolytic anemia*. Example 2: Loss of the dystrophin (an adaptor that links actin to plasma membrane) component of the membrane skeleton in skeletal muscle cells sensitizes these cells to contraction-induced damage, leading to *muscular dystrophy*.

What does Gelsolin do?

It is a calcium regulated actin severing protein. After cleaving the the filament, it caps the (+) end.

What is adducin?

It is a protein that *caps the barbed (+)* end of actin filaments in the membrane skeleton.

What is CAPZ?

It is a protein that caps the barbed (+) end of *actinomyocin* filaments.

What is macropinocytosis?

Macropinocytosis is the process by which a cell engulfs large amounts of fluid. As an example, dendritic cells of the immune system use macropinocytosis to sample potential antigens in their vicinity.

What does ARP2/3 do?

Nucleator of actin filament formation both de novo and from the sides of pre-existing filaments (side branching-characteristic 70 degree angle). The complex has a surface that resembles the barbed end of an actin proto-filament. This surface provides a template from whence new filaments grow. Important in the endocytosis process.

What are microvilli?

Microvilli - finger-like projections made from actin that commonly serve to *increase membrane area*

What is the structure of actin filaments (F-actin)?

Monomers are arranged into a *parallel two stranded helix with a right-handed twist*. Domains I and III of one actin molecule contact domains II and IV of the adjoining actin molecule. Domains III and IV mediate the contacts that hold the two protofilaments together. Domains I and II mediate most accessory protein contacts. Parallel fibers means that F-actin is polar. Barbed end = plus (+) end = fast polymerizing end. Pointed end = minus (-) end = slowly polymerizing end.

What are myosin?

Myosins are motor molecules that use ATP to pull on actin. Polymers of myosin in muscle cells are termed thick filaments.

What is the structure of myosin? What is the role of each structure feature? How is the processivity of myosin?

Myosins are the large (heavy) subunits of macromolecular machines that use ATP to move along actin filaments. a. Head domain = motor domain b. Neck domain = lever arm, binds light chains, length of neck + number of light chains bound sets step size c. Tail domain = cargo-binding/dimerization domain All myosin move along actin from the pointed end toward the barbed end. Nearly all myosin lack processivity, meaning their work cycles involved release from actin.

Describe the tropomyosin/troponin system.

Normally, tropomyosin/troponin cover the myosin head such that it cannot bind to the actin filaments. However, if calcium concentration is high enough, *Ca will bind to troponin*. After a conformational change, tropomyosin leaves myosin, leaving an exposed head. Myosin binds to actin to initiate contraction.

What is phagocytosis?

Phagocytosis is the process by which a cell engulfs and swallows large moieties in their environment. As an example, neutrophils remove bacteria from the interstitial spaces of our bodies via phagocytosis.

What are pseudopodia?

Pseudopodia - sheet-like extensions made from actin involved in *phagocytosis and macropinocytosis.*

What are stress fibers?

Stress fibers are *actinomyosin filaments of non-muscle cells*. Cells use these fibers to oppose persistent forces that impinge upon the cell and to mediate contractions involved in cell migration.

What is the terminal web? What is its structural function?

Terminal web is the especially thick membrane skeleton that is present under the *apical surface of epithelial cells*. It provides *structural support for adherens junctions* and is the foundation for actin-rich extensions. *Support role of F-actin.*

What are tropomodulins?

The only known family of *pointed end (-) capping proteins*.

How do vesicles generated from endocytosis get moved?

The polymerization of these new actin filaments generates a force, which helps drive these vesicles away from the plasma membrane and through the cortical skeleton.

What do WASP/WAVE proteins do?

When inactive, these proteins are bound to ARP2/3. When activated by small G-proteins, WASP/WAVE relieves *negative regulation of Arp2/3* and activates an actin recruiting function.

What diseases do mutations in WASP cause?

Wiskott-Aldrich Syndrome, a rare X-linked disease that is characterized by lack of platelets and dysfunctional T-cells.

What does profilin do?

a. Promotes actin polymerization. b. Is normally bound in an inactive state at the plasma membrane. c. When activated, profilin displaces thymosin through an *interaction with the barbed (+) end of G-actin.* d. Because profilin *does not bind to the pointed (-) end, profilin-bound actin can add to the barbed end of actin filaments.* e. Association of profilin-bound actin with the barbed end of a filament causes profilin to fall off actin. f. Because profilin displaces thymosin and does not interfere with actin interaction with the barbed end, *profilin effectively increases the pool of actin* that can polymerize into filaments, thereby driving actin polymerization.

What are stereocilia?

longer versions of microvilli, have specialized functions in the inner ear

What are lamellipodia?

sheet-like extensions made from actin that allow the cell to *flow in a given direction*

What are α-actinin?

α-actinin is an *extensible protein* with an actin-binding site at its N-terminus. Forms an antiparallel dimer, which positions an actin binding site at either end of the dimer. Crosslinks actin filaments together. Is located at the *Z-lines* of skeletal and cardiac muscle and in the *dense bodies* of smooth muscle. In non-muscle cells, it is localized in actin rich specializations of the plasma membrane such as microvilli, stereocilia and focal adhesions.