What is actin ? actin , a biopolymer, is found in the cytoskeleton. The sperical G-actin moleculuespolymerise to from F-Actin filaments which bundle together.CSKmechnics is dominated by actin bundle netwroks. It has a complictaed stress-strain respoonse, The cell soften if the strain was transient becoming fluid like.
One of the oldest polymer , exist on Eukaryotic cells,Mechanical Modulation Physical properties of tissues and cells change with disease For exampleCancer whichkills more than 7 million per and one way study Cancer metastisis as function of cells stiffness. Too low stiffnes can akecancrous cells squeese through the body easily Malaria which killls more than 1 million a yearMalrai infected cells are stiffer than health ones which make then un able to squeeze through blood capiliaries
Human walk differently on different substrates : sand, paved, gel. The same for cells, particularly adherent cells. Substrate stiffness influnce adhesion strucutre, cell dynamics and sytoskeletonassmply and orgniation and cell spreading. Dynamic adhesion on soft substrate , static focal adhesion on stiffsubstrate. Fig.2 Substrate stiffness influences adhesion structures and dy- namics (14), cytoskeleton assembly and cell spreading (17, 42), and differentiation processes such as striation of myotubes (28). (Top) The arrows point to dynamic adhesions on soft gels and static, focal adhesions on stiff gels. [Adapted from (14)] (Middle) The actin cyto- skeleton. (Bottom) A cell-on-cell layering in which the lower layer is attached first to glass so that the upper layer, which fuses from myoblasts that are added later, perceives a soft, cellular substrate.Cell recognize the underlying substrate composition Cells need to adhere to a solid to function. “Anchorage dependance “ they are not viable when suspended in fluid The mechanical response of cells is dependent on the substrate and the whole extracceullular matrix
In actin solution droplets, Huber et al showed that increased ion concentration led to highly ordered and regularly spaced actin bundle networks without the need for molecular motors. The same phenomena should happen to any volume decreased cell, on any substrate. We compress the cell by increasing the osmotic pressure of the cell’s medium by adding PEG to the solution. Fig. 1 Experimental procedure. Using a glass micropipette chilled G-actin solution is transferred onto a passivated glass substrate which is covered by a layer of hexadecane or silicone oil (A). Actin is allowed to polymerize and reach steady state conditions at room temperature (B). By removing some of the oil, evaporation from the droplet through the remaining oil layer is accelerated (C). When reaching the bundling threshold of the multivalent ion concentration an extended network of actin bundles appears throughout the whole droplet within minutes (E). The droplet volume can be monitored over time based on confocal images (D, squares) giving access to actual actin and ion concentrations (D, circles). In addition to a direct visualization (E), the intensity deviation of the fluorescence signal (D, triangles) presents a measure for the transition from F-actin to bundle networks
Images clearly shos less dense actin filaments on gel relative to glassWhen we compress the cells, by putting 10% peg into the soltuion leaving for about 10 minutes , we also note that
It is known that sustained pressure, increases cells stiffness. This may induce actin bundles network formation. The mehanisim for stiffness migght be , also the volume decrease increase the concentration Universal physical responses to stretch in the living cell . Trepat et al. Nature. Vol 447, 31 May 2007.
Ask for better ideas on how to quantify actin bundles
Ask for better ideas on how to quantify actin bundles
Compression induced formation ofActin bundle networks in Adherent Cells Burhan Saif Addin Ming Guo, Weitz Lab Harvard University ES 298r Mar 23, 2012
Outline• Introduction and Motivation• Background Information• Project Idea• Results and Discussion• Conclusion 1
Introduction• What is the Cytoskeleton (CSK) : – 3D protein scaffold. en.wikipedia.org/wiki/Cytoskeleton – Determine structure, shape, organization, .... – More than 8% of Human Genome encodes for cytoskeleton – Cytoskeleton mechanics are not well understood and the search for universal laws , if any, is in progress. – Contain three major skeletal proteins: actin, intermediate filaments, and microtubules.• What is role of Actin Filaments : – Cell motility and adhesion – Cell mechanical response and modulation. 2
Motivation: Why study cells actin networks?• How life evolved ?• Morphogenesis ?• How cytoskeleton work ? Cell Motility, etc ?• How Cells sense and process information ?• To understand Cell Mechanics and stiffness: – Physics not well understood. Are there universal laws ? – Understand Diseases. For example: Malaria and Cancer. – Cells signaling and gene expressions. 3
Adherent Cells feel and respond to the stiffness of their substrate Esoft~ 1kPa Estiff~ 30-100kPa Stress versus strain illustrated for different soft tissuesMechanism for Cellular response to substrate stiffnessare not well understood. Possible Mechanisms: -Molecular pathways. -Volume change ? 4“Tissue Cells Feel and Respond to the Stiffness of Their Substrate” Discher et al, Science, 18 Nov 2005.
How do we know that reducing the volume of the cell induce actin formation ? • In actin solution droplets, Huber et al showed that increased ion concentration and actin concentration led to highly ordered and regularly spaced actin bundle networks without the need for crosslinkers or motors. • In this work, we compress the cell by increasing the osmotic pressure of the cell’s medium by adding PEG (suck water out of cell) to the solution.Kas et al. Biophysics Society Meeting 2012, San Diego.“Counterion Induced formation of regular actin bundle networks”. Hubber et al. Soft Materials. March 2012 5
Cells on Glass without compression 6Images are by confocal fluorescence microscopy
Compressed Cells on Gel (10% PEG solution)Volume decrease might be the mechanism that induce actin networksformation thus cells stiffness. 8
Green line Actin’s Dye Fluorescence Intensity across the lines inis ~31 μm) the left figure which are cell’s cross-sections. . Rough Results: Without compression: 0.28 peaks/um With compression: 1.72 peaks/um 9
Possible Problems with this method of dye intensity quantization• Reproducibility.• Highly dense Interlinked actin filaments.• Unlabeled actin filaments might not be uniform*.*Polar patterns of driven filaments. Nature. VolkerSchaller et al. 24 June 2010. 10
Other method for quantifying actin bundles networks ?We attempted to quantifythe Actin based on intensity.How do you quantify this ? “Counterion Induced formation of regular actin bundle 11 networks”. Hubber et al. Soft Materials. March 2012
Conclusion• Volume change in cell can change Actin concentration or ion concentration, and hence induce actin polymerization.• Perhaps the substrate stiffness effect is not the only reason for actin polymerization.• Is actin polymerization due to volume change or due substrate or chemical signaling ? This remain to be investigated. 12
Acknowledgment• Ming Guo• Weitz Lab Members• Prof. David Weitz 13