1. Cytoskeleton Inside the plasma membrane is the cytoplasm. For a long time, it is believed that cytoplasm contains many organelles floating in a soluble compartment called cytosol. Besides the organelles, cytoplasm lacks defined structure and is a sac of thick water solution with proteins etc in it. This view has completely changed since the discovery of cytoskeleton. This is highly ordered “highway” system inside the cytoplasm that connects subcellular regions and coordinate communication inside the cell. 1.Three types of cytoskeleton filaments Microtubules, actin filament, and intermediate filament Principles governing the building of these structures: a)Each type is built by subunit proteins (tubulin, actin, lamin etc) b)Each type extend or shrink by subunit (de)polymerization. c)All types can interact and weave a complex network that hold the cell interior into a single entity. Such network can be extracted by detergent treatment.

2. A cytoskeleton meshwork extracted from a carrot Suspension cell after Cell wall digestion and detergent treatment (to solublize membranes).

3. 2. Microtubule (MT) and Actin filament (AF) 1)micro-anatomy: MT is a hollow tubule of 25 nm in diameter and can be many um long.

4. AF is a thread of 8 nm in diameter.

5. 2) The subunits: MT is made of tubulin proteins: two major types alpha and beta tubulin. AF is made of actin protein (single type). Tubulin is a GTPase and actin an ATPase—hydrolyze N (A or G) TP. Cytoskeleton subunit proteins are a group of most conserved proteins among all eukaryotes from yeast, plants, to human—very similar in sequence and structure. So the cytoskeleton in all these cells would be very similar. The genes for these proteins: often found in large families—why? 3) Dynamics of MT and AF a)Polymerization of tubulins and actins into the MT/AF: spontaneous process and can be produced in the test tube starting with subunits! Tubulin +GTP (or actin+ATP) -----  MT (or AF) Nucleation  elongation  steady state (depending on the critical concentration of the subunits, at which equilibrium is achieved)

7. b) Polarity of the MT/AF: The more active end is called “+” end and the other is called “-” end. Such polarity of MT/AF is caused by the intrinsic polarity of the subunit molecules, tubulin and actin. The tubulins always work together as dimers (alpha and beta are different protein with different property). Even the same actin molecule has polarity because the structure of one side is different from the other side.

9. Tubulin and actin are both NTPases. POlymerization activity Is determined by this activity—the more active the GTPase (tubulin) or ATPase (actin), the less active the polymerization. When GTP/ATP accumulate at the end (called GTP/ATP cap), it favors polymerization but GDP/ADP end Would favor depoly…

11. 3. MT/AF associated proteins: Associate proteins for regulating the dynamics of cytoskeleton And Cytoskeleton motors. Examples of MAP (MT-Associated Proteins) and ABP (Actin-Associated Proteins) to demonstrate the structure and function of these proteins 1) ABPs for regulation of actin filament dynamics: profilin (pollen allergen) binds actin monomers and lower the [actin] thereby inhibiting actin polymerization. Actinin and fimbrin—crosslink the actin filament to form network. AFs Actinin (larger than fimbrin and produces a larger crosslink space) fimbrin

12. Gelslin and high [ca2+] break the actin filament network by severing the filament. 2) Cytoskeleton motors: Three major types: myosin, kinesin and dynein. ---actin motors include two types of myosins---interaction of actin-myosin type II is involved in muscle activity and explains how the muscle produces mechanical force. Myosin type I is cytoplasmic myosin and involved in organelle transport and cytoplasmic streaming. Kinesin and dynein are Microtubule motors, They travel along the MT Highway. Their EM pictures were produced by freeze-etch rotary shadowing dyneins Myosin II kinesin

13. Myosin II often work in multiple subunit complex to slide the AFs and generate force. They travel from - to + end of AFs Myosin I can also work to slide Afs but most often totransport organelles along the Afs. They travel from + to – end.

14. Kinesins travel from – to + Dyneins travel in opposite Direction All motors consist of heavy and light chains/subunits. *The globular heads of all Heavy chains are ATPases. And the light chain tails are cargo specifying Devise—recognize the cargo They carry!

15. 4. Functions of cytoskeleton 1)Cell shape and growth: cell wall organization is largely determined by cytoskeleton; root hair, pollen tube tip growth (actin organization has key role) 2)Cellular movement: cytoplasmic streaming (actin-myosin), organelle movement and transport, vesicle transport and fusion 3)Cellular organization and communication 4)Cell division! (guest lecture on how cytoskeleton participate in cell division—exciting pictures and movies recording the processes occuring inside the cell) 5)cell signaling

17. Kinase inhibitor disrupt MT organization

18. Interaction between MT/PM/Wall