Dissecting the complexity of cell structure 2 microfilaments
Download
1 / 46

Dissecting the Complexity of Cell Structure 2: Microfilaments - PowerPoint PPT Presentation


  • 122 Views
  • Uploaded on

Dissecting the Complexity of Cell Structure 2: Microfilaments. How actin-based polymers contribute to cellular structure, behavior, and motility. An overview of the actin cytoskeleton in a cultured epithelial cell, using antibodies and immuno- fluorescence.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Dissecting the Complexity of Cell Structure 2: Microfilaments' - hiroko


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Dissecting the complexity of cell structure 2 microfilaments

Dissecting the Complexity of Cell Structure 2: Microfilaments

How actin-based polymers contribute to cellular structure, behavior, and motility


An overview of Microfilaments

the actin

cytoskeleton in

a cultured

epithelial cell,

using antibodies

and immuno-

fluorescence.

Actin is at the cell cortex and also organized into fibers that extend through the cell.


Formation of microfilaments (MFs) from Microfilaments

the protein actin


Actin polymerization is controlled, in part, by an Microfilaments

initiating complex that includes actin-like proteins


The Arp2/3 complex not only initiates MF Microfilaments

polymerization, it can bind to the walls of

existing MFs, promoting the formation of

branches, which turn that region of the cell

into a gel (as opposed to a “sol”).


Actin at the Microfilaments

edge of a

cultured

cell, looking

at all actin

(top) or only

recently

added

actin

(bottom).

Actin

polymerizes

at the cell periphery



Actin polymerization membrane-end of MFs

is also regulated by

small proteins that

bind to soluble actin

and modify its

behavior in solution.

Here is profilin, which

catalyzes the exchange

of ADP for ATP in the

actin cleft, increasing

the pool of polymer-

ization-competent

monomer


There are also proteins that retard MF growth: membrane-end of MFs

While profilin

enhances MF

growth by

increasing

the concentration

of ATP-actin,

Thymosin reduces

it by making a

complex that will

not polymerize


Diagramatic representation of the pathways membrane-end of MFs

The help to regulate MF formation


So how does a complex process like this actually work
So how does a complex process like this actually work? membrane-end of MFs

  • Amoeboid motility does not require MTs: it proceeds normally in the presence of MT poisons

  • It is immediately poisoned, however, by drugs that block actin polymerization, like Cytochalesins D and E or Latrunculin A

  • It fails in several mutant strains that lack key actin assembly proteins (though it is amazingly robust to mutation)

  • Infer, to understand amoeboid movement and related kinds of cell motion we need to understand the control of actin polymerization and organization


Proteins that bind to the walls of MFs can bind membrane-end of MFs

them together in different geometries


Four proteins that bind the sides of MFs and membrane-end of MFs

bundle them into different geometries



TEM image and diagram of MF membrane-end of MFs

Bundling in a microvillus


Diagram of MF web formed when cross-linking membrane-end of MFs

is done by the long, fibrous protein, Filamin


When filamin is under-expressed, due to mutation, cell morphology and motility are abnormal. Cells depleted for Cell expressingfilamin by LOF mutation normal filamin levels


Like MTs, MFs can bind some associated morphology and motility are abnormal.

proteins that alter the properties of the polymer.

Cofilin binds to F-actin and distorts the polymer,

making it less stable.


Gelsolin also bind the MF wall, distorting is and morphology and motility are abnormal.

inducing breaks that shorten the average fiber.


Gelsolin and morphology and motility are abnormal.

proteins like it

can break up MF

either in vitro (as

shown here) or

in vivo, leading to

rapid reshaping of

the actin

cytoskeleton


Breaks in MFs mean both more and shorter morphology and motility are abnormal.

MFs therefore more ends (for a given amount

of polymer). Thus, both growth and shrinkage

of polymer can be faster after MF severing.


Capping proteins, like Cap-Z can silence one morphology and motility are abnormal.

MF end for further subunit addition; in this case

it is the fast-growing (plus) end that is

turned off.


All these processes can be regulated to make morphology and motility are abnormal.

For a quite complex behavior of the MFs in vivo.

Blood platelets (thrombocytes) looking as they

do in circulating blood (left), after attachment

to a surface (center) and during a platelet

“reaction”, where they attach to a substratum

and contract. All this is MF assembly-dependent.


But What Initiates MF morphology and motility are abnormal.

Polymerization

at the PM?


Projection morphology and other actin-dependent shape changes suggest that controlled polymerization can also give rise to unidirectional fibers, the filopodia

  • Microvilli are of this form

  • Dynamic projections from not only platelets but also neuronal growth cones are of this form

  • Even projections from sperm and egg, involved in fertilization, are of this form

  • There must be a membrane-associated actin initiator that does not involve branching


Formins are now recognized as molecules that can initiate MF polymerization at the PM and allow continued addition of actin at the PM while an association with the membrane is maintained

  • Formins contain conserved domains, the Formin-homology (FH) domains. FH1 and 2 define a comparatively short polypeptide that can serve as an efficient nucleator of MF polymerization

  • Formin-initiated polymerization adds actin subunits AT the PM, pushing the already formed MF away

  • This allows PM-controlled events to regulate aspects of MF organization, much as MTOCs control MT formation


Movements of PM-associated “cortical” actin can affect the position and organization of other components of cytoplasm, like MTs



Actin “stress fibers” are initiated by and attached to the cell cortex.Cell margin seen with The same region seen withoptics that show proximity fluorescence optics and anbetween the cell’s surface antibody that lights up actin

and the underlying substratum

These are “focal adhestions” that mark site of cell attachment


Another view of stress fibers (green) and “focal adhesions”, stained with antibodies to vinculin, a component of stress fiber adhesion sites


There are specific trans-membrane proteins and adaptor proteins that connect MFs with the extracellular milieu


To promote tissue strength, cadherins are specifically but non-covalently bound to the MFs of the cytoskeleton. The linking proteins include catenins, which play a role in signaling between tissues.


Cadherins bind the MF cytoskeleton of one cell to that of its neighbors, forming a mechanical

unit. This coupling contributes to the mechanical integrity of a tissue.


In some cells, cadherins and actin MFs form ordered arrays that can work like a contractile ring and control the cell’s diameter.


Integrins are membrane proteins that bind cells that can work like a contractile ring and control the cell’s diameter.

to the “Extra-Cellular Matrix” (ECM). The integrins make bonds between the actin cytoskeleton and the fibers of the ECM, such as collagen and fibronectin.


Transmembrane proteins link the actin-dependent cytoplasmic motility that pulls on MTs with extracellular material, such as beads that can attach to the cell’s surface


Thus, we can recognize a mechanical continuum that runs from cytoplasmic MFs, controlled in part by MTs, through the plasma membrane to the molecules of the extracellular matrix (ECM).

  • The questions defined by this idea include the identity and behavior of the linker molecules (cadherins, integrins, and others) and the processes and molecules that control the behavior of the fibers and their states of polymerization and linkage



Reminder of the mechanism for signaling by small G-proteins, like Rac, Rho, and CDC-42: GTP-bound state is active and turns on down-stream proteins. GAPs and GEFs control the relative concentrations of GTP- and GDP-bound forms of the protein


In budding yeast there is a signaling cascade that links a pheremone to regulation of the actin cytoskeleton

Discovery of such control pathways has motivated a

Search for homologous paths in other cell types


There are >8 down-stream targets for each of the small G-proteins involved in regulation of the actin cytoskeleton

  • Rho-GTP leads to the activation of cytoplasmic myosin, which in turn promotes actomyosin-dependent structures, like stress-fibers

  • Rac-GTP activates WASp, which in turn activates the Arp2/3 complex, and it also promotes uncapping of MFs by removing CapZ or Gelsolin, inducing extensive, branched +-end MF growth

  • CDC42-GTP also activates the WASp protein but Formin is also activated, promoting MF assembly into membrane-associated bundles


A plausible mechanism for the organization of actin in the cell’s cortex and for its role in extension of a lamellipod


Signal transduction cascades that activate small G-proteins have now been found in a wide range of cells


Functional Rac is important for mebrane-associated actin’s response to MT invasion of the cell cortex



ad