Lecture Outline: Actin Motors and Microfilaments Fall 2024

Actin motors -Myosins are motors that move along F-actin -move towards the (+) or barbed end -use ATP to drive movement -2 classes of myosins: 1. “Conventional” myosins ex: Myosin II 2. “Unconventional” myosins -at least 17 different classes identified...

Actin motors -Myosins are motors that move along F-actin -move towards the (+) or barbed end -use ATP to drive movement -2 classes of myosins: 1. “Conventional” myosins ex: Myosin II 2. “Unconventional” myosins -at least 17 different classes identified ex: Myosin V  Actin motors: Myosin II structure -Composed of: - 1 pair of heavy chains - 2 pairs of light chains -Heavy chain has “head domain” -ATP-hydrolysis domain = power generation -actin binding region Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Figure 16.21  Actin motors: Myosin II structure -Composed of: - 1 pair of heavy chains - 2 pairs of light chains -Heavy chain has “head domain” -ATP-hydrolysis domain - power generation -actin binding region -Heavy chains coil together to form a “tail” -allows the protein to form filaments -called “bipolar filaments” -staggered arrangement of myosin molecules such that their tails point inwards and heads point out  Figure 16.22B Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall.  Actin motors: Myosin II function -present in several cell types; especially in muscle -in most cells, myosin II is required for cell motility and cytokinesis during cell division -muscle cells require myosin II for muscle contraction  Muscle contraction: Myosin II function Muscle cells: very long, cylindrical cells with hundreds of nuclei – actually called muscle fibres -formed from fusion of several smaller cells -Muscle fibres are a collection of myofibrils -Myofibrils are composed of organized repeating structures: sarcomeres Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Muscle contraction: Myosin II function -Sarcomeres are made up of thick and thin filaments with regions of overlap between them: the contractile units of muscle -Thin filaments = microfilaments (actin filaments) -Thick filaments = myosin II bipolar filaments -Gives muscles a striped or striated appearance Figure 17-40 Essential Cell Biology (© Garland Science 2010) Actin + ends are attached to the Z discs. + end + end Figure 16.27 C,D Myosin II function: muscle contraction -The multiple myosin heads bound to actin use energy from ATP hydrolysis to move the actin (thin) filaments inwards -sarcomere contracts = muscle contracts Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Myosin generates force by coupling ATP hydrolysis to conformational changes Figure 16.24 Organization of accessory proteins in a sarcomere Figure 16.29 Titin is elastic and changes length as the sarcomere contracts and relaxes  Figure 16.31 A Tropomyosin interferes with the binding of actin to the myosin heads. Troponin, a Ca2+ binding protein, is attached to tropomyosin. When Ca2+ ions are present and bind to troponin, it changes the conformation of tropomyosin, which moves away from actin filament, allowing the myosin heads to make contact with the actin filament. This can then allow muscle contraction.  Unconventional myosins Unconventioanl myosins are involved in: -intracellular transport -microvilli formation -do not form filaments -Myosin V is an example: -contains 2 heads like myosin II -has a very long ‘neck’ region -allows this myosin to make long strides on actin -functions in intracellular transport -head binds actin -tail binds vesicles/cargo -uses ATP to move cargo to (+) end Myosin V ‘walking’ along actin ‘Hand-over-hand model’ Figure 16.36 A 36nm steps à very long neck region  Unconventional myosins Myosin V function -carries many kinds of vesicular cargo -the cargo travelling to the synapse regions (vesicles) are often transferred from MT motors to Myosin V -mediates partitioning of organelles (mitochondria, peroxisomes) to daughter cells  cargo Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall.  Microfilaments: cell migration -example of non-muscle motility of cells -is essential for: -development of an embryo -formation of blood vessels -wound healing -spread of cancerous tumours Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall.  Microfilaments: cell migration A series of repetitive actions are performed: 1. A part of the cell protrudes in the direction that the cell wants to move = e.g.“lamellipodium” 2. The cell protrusions attach to the surface beneath 3. The bulk of the cell is pulled forward towards those surface contacts It is now ready to go through these series of movements again Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Different cells generate different types of protrusive structures including lamellipodia -the cell protrusion and forward pulling forces are generated by actin polymerization -forces for the retraction of the rear of the cell is generated by myosin II motors  Microfilaments: cell migration Directed cell motility: -a stimulus is received at one end of the cell –PM receptors bind proteins -a family of proteins in the cytosol become activated = WASP proteins -activated WASP proteins then activate Arp2/3 complex proteins -activated Arp2/3 proteins serve as the nucleating site for new actin filaments -new actin filaments form -activated Arp2/3 proteins bind to these new actin filaments and nucleate more actin filaments (branches) -the growing barbed (+) ends push the PM outward in the direction of the stimulus -as newer actin filaments form, the older filaments are disassembled by proteins such as cofilin Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Bacteria can hijack the host actin cytoskeleton Listeria monocytogenes The ActA protein on the bacterial surface activates the Arp2/3 complex to nucleate new filament assembly along the sides of existing filaments. Filaments grow at their plus end until capped by capping protein. Actin is recycled through the action of cofilin, which enhances depolymerization at the minus ends of the filaments. By this mechanism, polymerization is focused at the rear surface of the bacterium, propelling it forward. Figure 16.17C  Microfilaments and pathogens -The ActA protein on the bacterium recruits Arp2/3 to build actin filaments -Use these actin “tails” to push themselves forward in the cytoplasm -Actin comet tails can be seen in infected cells THE CYTOSKELETON: Microtubules  Microtubules hollow, tubular structures found in almost all eukaryotic cells -25nm diameter -the building blocks are made up of a protein known as tubulin -tubulin subunits come in two forms: α and β - α and β tubulin are very similar in their structure and fit together very well - form dimers = heterodimers Figure 16.37A,B  Microtubules: protofilaments -These α-β tubulin dimers are the building blocks of a long polymer known as a protofilament =head to tail -The protofilament is asymmetric: α- tubulin on one end and β-tubulin on β end the other end = protofilament has polarity -α-tubulin end of the protofilament is called the minus (-) end and the β- tubulin end is called the plus (+) end α end  Microtubules α-tubulin has a bound GTP – does not get hydrolyzed or exchanged -physically trapped at the dimer interface β-tubulin also has a bound GTP -this bound GTP is slowly hydrolyzed to GDP sometime after polymerization occurs -β-tubulin is a GTPase Figure 16.37A,B Adding heterodimers to the plus end  Microtubules -Each microtubule is composed of 13 protofilaments put together -protofilaments are aligned side by side in a circular pattern -held together by non-covalent interactions --hollow core -each protofilament in a microtubule has the same polarity = microtubule has polarity Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall.  -hollow core heterodimer microtubule protofilament  Microtubules (MTs) : polarity -The “(–)-end” is anchored in the centrosome (perinuclear region) -polymerization begins from here -referred to as an MTOC = microtubule organizing centre -starts with a type of tubulin called γ- tubulin at MTOC -Centrosome = pair of centrioles + proteins -The “(+)-end” extends out from the MTOC towards the plasma membrane (cell periphery) MTOC Figure 16.41C Gamma-TURC  -depolymerize microtubules with a drug = nocodazole -MTs reassembled from the MTOC out towards the PM -must start assembly at MTOC/centrosome Figure 16.44  Microtubules: dynamic instability -MTs can assemble and disassemble rapidly -turnover -Growing and shrinking microtubules coexist in the cell -This rapid growth and shrinkage is referred to as “dynamic instability”  Microtubules: dynamic instability -Recall that β-tubulin is in the GTP bound state when microtubules are assembling -Sometime after polymerization, GTP is hydrolyzed to GDP -GDP-tubulin is prone to depolymerization Figure 16.40A -GTP hydrolysis is occurring usually at a slower rate than addition of new GTP- tubulin dimers on the (+) end. The GDP form of β-tubulin in the middle of a microtubule cannot spontaneously pop out -The GDP form of β-tubulin is unstable when present at the very end of a microtubule's growing (+) end. -When GTP hydrolysis catches up to the tip of a microtubule, the structure will depolymerize =catastrophe Figure 16.40 B Microtubule-binding proteins modulate filament dynamics and organization Panel 16.4  Microtubules: dynamic instability -the stability of microtubules can be increased or decreased -Ex: binding of certain proteins 1. Microtubule associated-proteins (MAPs) -typically increase stability -MAPs have 2 domains: one that binds the microtubule and one that extends as a filament -phosphorylation of MAP2 controls its binding ability = can’t bind if phosphorylated Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. Figure 16.46  Microtubules: dynamic instability 2. Protein that bind to the + end = + end tracking proteins = +TIPs Growth and shrinkage of MTs can be controlled by +TIPs such as kinesin-13 and XMAP215 Figure 16.48  Microtubules: dynamic instability 3. Tubulin- sequestering proteins = stathmin Modulate microtubule dynamics Figure 16.50  Microtubules: dynamic instability 4. Microtubule-severing proteins = katanin Modulate microtubule dynamics Figure 16.51C  Microtubules: dynamic instability -cells typically have both stable and dynamic microtubules -Ex: stable microtubules involved in positioning of organelles -Ex: dynamic microtubules aid in cell motility  Microtubule motor proteins -multi-protein complexes -motor proteins convert chemical energy (ATP) to mechanical energy -are ATPases -composed of 2 domains: one binds the microtubule, the other binds the cargo -help transport cargo along microtubule tracks Examples of cargo: mitochondria, chromosomes, vesicles -motor proteins move unidirectionally along microtubules -move according to polarity of the MTs  Microtubule motor proteins 2 types of microtubules motors: _ + 1. Dynein – moves to the “(-) end” of microtubules = retrograde 2. Kinesin – moves to the “(+) end” of microtubules= anterograde  MT motors: Kinesins Part of a large superfamily – humans have 45 different kinesins! “Conventional” kinesin Neck region different; travels to the minus end! Figure 16.52  MT motors: Conventional Kinesin (kinesin 1) - (+)-end directed MT motor protein -a tetramer protein -2 identical heavy chains & 2 identical light chains -4 structural features: 1. Heads: ATPase – binds MTs 2. Neck – determines direction of movement (i.e. towards (+) end) 3. Stalk –provides flexibility or movement of heads and tail 4. Tail – binds cargo Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall.  Adaptor proteins: tend to be integral or peripheral membrane proteins that are part of the cargo. Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall. -A “hand-over –hand” mechanism of movement along MTs -single “step” = 8nm * *dependent on ATP -can move long distances on MTs without falling off = processive Figure 16.53 Microtubule motors: Cytoplasmic Dynein 1 -a very large protein complex composed of 2 “heavy chains” -several “intermediate “ and “light” chains -large head domain = ATPase - force generation -stalk domain = MT- binding domain Figure 16.54B -does not interact directly with cargo -uses an adaptor protein and dynactin to bind cargo Dynactin has components that: 1. Bind weakly to MTs 2. Bind to dynein 3. Include actin-like filaments that associate with cargo 4. Dynactin associates with two molecules of cytoplasmic dynein as well as with an adaptor protein that mediates the connection to a cargo. Figure 16.55  Microtubule motors: Functions Dynein: “(-)-end” directed MT motor -important for spindle positioning and chromosome separation in mitosis -moves various organelles like Golgi, lysosomes to their perinuclear position -move endosomes to cell interior -also move various vesicles in the cytosol Kinesin: “(+)-end” directed MT motor -move organelles such as peroxisomes and mitochondria towards the outside of the cell -moves secretory vesicles to the cell periphery  Microtubules: the mitotic spindle Green = MTs Blue = DNA (chromosomes) Red = point of attachment of MTs to DNA pole pole (-) end of MTs at spindle poles (+) end of MTs attached to DNA MTs slowly disassemble (at + end) Dynein moves DNA to (-) end of MTs http://people.virginia.edu/~djb6t/LabWeb/photos/Anne2.jpg -towards poles -cell division occurs  Mutant analyses Normal motor protein Mutant motor protein -pigment granules do not reach their proper destination if the motor protein that carries them there is mutant -no transport to cell periphery Karp’s Cell and Molecular Biology: Concepts and Experiments.” 8th edition. Janet Iwasa & Wallace Marshall.