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Cell Behaviour 3 – Cytoskeleton and Cell Motility Anil Chopra 1. 2. 3. 4. Describe the types of movements that cells undergo. Explain treadmilling in the context of filament polymerisation Describe what is meant by molecular motors Describe the movement of vesicles along microtubules, with reference to the molecular motors. 5. Describe myosin molecules in general, and the molecular organisation of myosin II. 6. Describe the formation of myosin filaments. 7. Describe the structure of a skeletal muscle cell, and the arrangement of filaments and membranes within it. 8. Summarise the sliding filament mechanism of contraction of striated muscle. 9. Describe the sequence of enzymatic changes making up the cross-bridge cycle of muscle contraction. 10. Explain the molecular basis for muscle stiffness (rigor mortis) after death. 11. Describe the genetic of myosin heavy chains and the relationship between fibre type and myosin heavy chain expression 12. Describe the functional difference between muscle cells expressing myosin heavy chains I and II. 13. Describe the distribution of fibre types in human skeletal muscle, and the effect of sporting activity on fibre type distribution. 14. Compare the mechanisms of control of the cross-bridge cycle in striated and smooth muscle. 15. Give examples of the different classes of myosin that have been identified, and correlate their structure with their different functions. Examples of cell movement Migration of phagocytic cells towards site of infection Migration of cancerous cells away from site of primary tumour - invasion Migration of cells during embryological development Cytoplasmic streaming Muscle contraction Swimming: waving of cilia/flagellae; movement of liquids Transport of organelles, Movement of vesicles Phagocytosis Mechanisms of Cellular Movement • Involves coordinated shape changes due to cytoskeleton (actin mainly) • Needs appropriate signalling to coordinate parts of cell and control direction • May depend on extracellular signals and receptor pathways • Cells become polarised - line up with a thin actin-containing extension (lamellipodium) forming the leading edge • Microtubule system also is aligned - MTOC (microtubule organising centre) is forward of the nucleus, as is the Golgi Cellular Motors Treadmilling This is where polymers move by polymerising at one end and depolymerising at the other. Kinesins Kinesins move vesicles along microtubules by “walking” along the microtubules toward the “plus” end. They hydrolyse ATP to ADP and Pi. Dynein and NCD Dynein and NCD (nonclaret disjunctional) move vesicles towards the minus end of microtubules. These also hydrolyse ATP to ADP and Pi. Myosin There are a number of different myosin classes each with their own function. The one in muscle is sarcomeric (class II). It is a heterohexamer: two identical heavy chains and two pairs of non-identical light chains, the essential and the regulatory light chains. Its head contains an actin binding domain and an ATPase. Its neck is helical and contains a sharp bend at the LMM-rod junction. Its tail consists of an α-helix 1096 dimerised residues. The myosin filaments in skeletal muscle are packed in such a way that the amount of contractile material in a given volume is increased to give maximal power. This makes it fast, powerful, efficient and able to be adapted by training. They are packed in a helical structure. Each bundle of filaments is a myofibril, of which there may be hundreds in each skeletal muscle cell. T-cells are invaginations in the sarcoplasmic membrane. The sarcoplasmic reticulum is a specialised endoplasmic reticulum, storing calcium A sarcomere is the repeating distance between adjacent Z-lines. H-band consists of myosin filaments (thick filaments) I-band consists of actin filaments (thin filaments) A-band consists of myosin filaments and thin filaments in the overlap region. Actin filaments are joined together at the Z-line. Myosin filaments are joined together at the M-line Sarcomeres get shorter when muscles contract. In this process they use ATP. It is brought about by the “cross-bridges” that form between the myosin and actin molecules. The heads of the myosin molecule bind to sites on the actin molecule when activated. This results in the filaments sliding over one another. In the absence of ATP, Actin and myosin are tightly bound, conferring rigidity and stiffness to the muscles. This is what happens after death and is called rigor mortis. The state is reversible upon addition of ATP into the myofilament space. Myosin Genes There are 8 heavy chain genes, 2 cardiac genes : MyHC-a – chromosome 14 MyHC-b also called MyHC type I or b-slow, chromosome 17 6 skeletal genes MyHC-embryonic – chromosome 17 MyHC-perinatal – chromosome 17 MyHC-IIa – chromosome 17 MyHC-IIb – chromosome 17 MyHC-IIx/d – chromosome 17 MyHC-extraocular – chromosome 14 Embryonic and perinatal mysosin heavy chains predominate during early skeletal muscle development and persists in some muscles such as extra-ocular, laryngeal and masseter. The cardiac MyHCs (of which there are 2 – MyHC α and β) are mainly expressed in the heart, however, MyHCα is also expressed in the masseter and extraocular muscles, and MyHCβ is expressed in skeletal muscle “slow type I” and “fast type IIa, IIb and IIx”. The relative types of myosin between species and within a species is constantly evolving slowly with different evolutionary advantages. Some muscles in the body are tonic (i.e. they have more than 50% MyHC I / MyHCβ fibres) or phasic (more than 50% MyHC II). The relative amounts of slow type I and fast type II fibres in muscles can be changed by training. Skeletal & Cardiac Muscle Contraction At the neuromuscular junction, the action potential crosses the synapse. The depolarisation of the muscle cells results in the release of Ca2+ from the sarcoplasmic reticulum. Ca2+ binds to troponin on tropomyosin causing it to change shape and expose the myosin binding sites on the actin. Myosin heads bind to the sites and cause the sliding chain. Smooth Muscle Contraction Controlled by the thick filaments, not the thin filaments as in skeletal or cardiac muscle. Calcium released into the cell as a result of electrical or hormonal activity at the cell surface binds to a soluble protein, calmodulin. The Ca-activated calmodulin binds to and activates myosin light chain kinase (MLCK) which phosphorylates a small protein that binds around the tail of the myosin head, myosin light chain 2 (Myosin-LC2). Phosphorylation of M-LC2 allows myosin binding to actin and contraction.