MCB 2210 L25: Interphase

Direction of MT motors

Kinesin → (+) end

Dynein → (-) end

Describe actin + MT + motor functions

Actin

• Membrane protrusion (lamellipodia + filopodia)

• Cell adhesion (stress fibers + focal adhesions)

• Endocytosis + trafficking (Clathrin pits + endosomes)

• Cytokinesis

Microtubules

• Organelle position (ER + Golgi)

• Anterograde transport (ER → Golgi → Plasma Membrane)

• Retrograde Transport (Endosome → Golgi → ER)

• Mitosis

What cell is commonly used to observe membrane protrusions?

What edge of the cell drives motion?

Which 2 actin-based structures are involved in membrane protrusions?

Fibroblast = cell of connective tissue

Leading edge = dynamic, forward-facing protrusive edge of migrating cell that drives forward motion

• Lamellipodia = thin sheet-like structures

• Filopodia = thin needle-like structures

  • Push plasma membrane forward by adhering to substrate or sweeping over top of cell as a ruffle

• Stress fibers = cell adhesion + contraction

How can the behavior of actin filaments of leading edge be observed?

Microinjecting cell w/ fluorescently labeled actin

• Fluorescent actin = incorporated into filaments @ extreme leading edge

• Demonstrates that actin polymerization is taking place @ leading edge

What drives membrane protrusion?

What describes the rearward movement of cytoskeleton?

Actin assembly → pushes membrane forward

• Energy from ATP hydrolysis by actin → drives process

Retrograde flow = continuous rearward movement of actin cytoskeleton

• Filaments pushed back by membrane resistance + myosin-driven contraction

• TREADMILLING

• Cofilin = actin severing protein → actin depolymerization @ leading edge

Describe what fluorescence speckle microscopy + what phenomenon it demonstrates

Fluorescence speckle microscopy = live-cell imaging technique using very low [fluorescently-labeled subunits] → “speckle” pattern

• Visualize retrograde flow

• Meshwork pushed backwards as membrane = moving outward

• Membrane → resistant

• Pushes meshwork inwards

Describe how F-actin is organized in protrusive structures

Barbed (+) ends → membrane (direction of protrusion)

• Lamellipodia = branched actin network

  • Nucleated by Arp2/3 complex + WASP protein

• Filopodia = tight parallel bundles of straight actin = finger-like protrusions

Describe actin assembly into stress fibers

Stress fibers = long bundles of actin filaments below cell surface

• Focal adhesions = where stress fibers terminate + attach to underlying substrates

• α-actinin = cross-linker proteins

• Myosin thick filament = bridge bundles together

• Integrins = transmembrane protein that connect actin → extracellular matrix

What triggers dynamic actin specialization?

What are the 3 types relevant to actin specialization?

Small G-proteins → Rho family GTPases

Rho family GTPases = members of Ras

• Rho, Rac, Cdc42

• Act as small molecular switches b/w:

  • Active form = bound to GTP

  • Inactive form = bound to GDP

GEFs = Guanine Nucleotide Exchange Factors = GDP → GTP = ACTIVATE

GAPs = GTPase Activating Proteins (GAPs) = GTP → GDP = DEACTIVATE

GDIs = Guanine Nucleotide Dissociation Inhibitors (GDIs) = stabilize GDP

What are the three types of pathways that activate WASPs and/or Formins and drive actin assembly in cells?

Rho → Formin → Stress fiber (straight tightly bundled actin)

Rac → Arp2/3 + WASP → Lamellipodia (branched actin)

Cdc42 → Formin + Arp2/3 + WASP → Filopodia (finger-like protrusions, spikes)

Explain an experimental method that can be used to create the 3 distinct actin structures.

1. Microinject GTPase of interest in dominant active form (constantly active) + observe actin forming structures

   1. Rho → stress fibers (tightly bundled straight actin)

   2. Rac → lamellipodia (branched actin)

   3. Cdc42 → filopodia (finger-like protrusions, spikes)

What 2 membrane trafficking processes does actin cytoskeleton direct?

1. Endocytosis

   1. Myosins → move on actin near cell surface

   2. Switch to MT motor-based movement on MTs

2. Endosome trafficking

   1. Endosome rocketing = endosomes polymerize actin on one side through cytoplasm by propelling fast actin polymerization

What type of actin assembly facilitates endocytic internalization?

Branched actin assembly → Arp2/3 complex, CapZ, WASP

How is actin assembly involved in membrane sorting in endosomes?

Actin assembly marks regions of membrane that need to be recycled back → different patches of membrane = recycled

Actin = markers to sort membrane into endosomes

What 2 processes of the secretory pathway are controlled by the MT skeleton?

• Organelle position

• Membrane transport

  • Ex. Anterograde transport (ER → Golgi → Plasma membrane

    • Use tracks of MTs + motor proteins → travel through compartments

Why are ER membranes dynamic?

ER membranes = connected to MTs

Explain Golgi and ER position in the cell in relation to motor proteins

How do drugs affect the position of the ER and Golgi?

MTs = help position Golgi + ER

• Golgi → centrosome

  • Dyneins help keep Golgi near centrosome

• ER → along MTs + cytoplasmic branches

  • Kinesins help keep ER positioned to cell periphery

Drug added → MT = disrupted → Golgi fragmented + ER collapses

What motor protein controls retrograde transport?

What motor protein control anterograde transport?

Dynein → (-) direction

• Golgi @ centrosomes (-) ends

Kinesins → (+) direction

• ER @ cell periphery (+) ends

What is COPII coat directly linked to?

Dynactin = intermediate = link b/w dynein + cargo

Explain melanosomes for organization look like for melanosome organization

A. What will melanosome organization look like if you treat cells with taxol?

B. What will melanosome organization look like if you treat cells with nocodazole?

C. What happens when you wash away the nozodazole or taxol?

A. Taxol → stabilizes MT → CANNOT depolymerize

B. Nocodazole → MT depolymerization

C. Regrowth of MTs → melanosomes = free to interact

How phosphorylates non-muscle II light chains?

Myosin Light Chain Kinase (MLCK) = phosphorylates Myosin II light chains → functional myosin thick filament complex assembly

• Inactive myosin = dephosphorylated

• Active myosin = phosphorylated