The document discusses a study examining how compressing cells by decreasing their volume through osmotic pressure induces the formation of actin bundle networks. The study compressed cells placed on a gel substrate by adding a polymer (PEG) to the solution, sucking water out of the cells. Confocal microscopy images show cells before compression have sparse actin filaments, while compressed cells exhibit dense, bundled actin networks. This suggests decreasing cell volume through compression may cause actin polymerization by increasing actin concentration or ion concentration within cells. However, other factors like substrate stiffness or chemical signaling could also influence actin polymerization, requiring further investigation. The document outlines the motivation, methods, results and conclusions of this study.
32 Ways a Digital Marketing Consultant Can Help Grow Your BusinessBarry Feldman
How can a digital marketing consultant help your business? In this resource we'll count the ways. 24 additional marketing resources are bundled for free.
It discuss about the cell, cell theory, Cell Theory Timeline,CELL DIVERSITY- unicellular, multicellular, size of cells, size of cells in human, shape of cell, structure of cell, Microscope, CELL CONSTITUENTS- cell wall/cell membrane, nucleus, cytoplasm in detail
6.Experiments with Xenopus (frog) in the late 1950s demonstrated tha.pdfwailesalekzydelore94
6.Experiments with Xenopus (frog) in the late 1950s demonstrated that cells within the 3 germ
layers had an affinity for each other and thus indicated that they had differentiated enough to be
“biased” toward a specific cell layer (endoderm, mesoderm, ectoderm). Describe an experiment
that demonstrated how the 3 different layers of the gastrula have an affinity for each other.
7.Morphogenesis involves the orchestration of Cell Affinity, Cell Adhesion, and Cell Migration.
How do the adhesive molecules play a role as cells of the embryo change their affinity for one
another?
PLEASE ANSWER BOTH QUESTIONS
Solution
1. Taking an advantage of discovery of amphibian tissues become dissociated into single cells
when placed in alkaline solupans,
2. The prepared single-cell suspensions from each of three germ layers of amphibian embryos
soon after the neural tube had formed.
3. Take two or more of these single-cell suspensions could be combined in various ways. When
the pH of the solution was normalized, the cells adhered to one another, forming aggregates on
agar-coated petri dishes.
4. This experiment shows affinity of 3 different layers of the gastrula.
Cell Affinity: a) The inner surface of the ectoderm has a positive affinity for mesodermal cells
and a negative affinity for the endoderm.
b) While the mesoderm has positive affinities for both ectodermal and endodermal cells.
c) Mimicry of normal embryonic structure by cell aggregates is also seen in the recombination of
epidermis and neural plate cells.
Cell Adhesion: a) cadherins are calcium-dependent adhesion molecules. They are critical for
establishing
and maintaining intercellular connections, and they appear to be crucial to the spatial
segregation of cell types and to the organization of animal form.
b) The cadherins are anchored inside the cell by a complex of proteins called catenins and the
cadherin-catenin complex forms the classic adherens junctions that help hold epithelial cells
together.
Cell Migration: a) migration involves the adhesion of the cell to its extracellular substrate. The
moving cell needs
something to push on, and attaches to the surrounding matrix.
b) Integrins span the cell membrane, connecting the extracellular matrix outside the cell to the
actin cytoskeleton on the inside of the cell. These connections of actin to integrin form focal
adhesions on the cell membrane where the membrane contacts the extracellular matrix.
c) Myosin and its regulators provide the motive force along these actin microfilaments.they are
linked with the lamellipodial actin at the sites of adhesion.
d) Release of adhesions in the rear, allowing the cell to migrate in the forward direction. It is
probable that stretch-sensitive calcium channels are opened and that the released calcium ions
activate proteases that destroy the focal adhesion sites..
32 Ways a Digital Marketing Consultant Can Help Grow Your BusinessBarry Feldman
How can a digital marketing consultant help your business? In this resource we'll count the ways. 24 additional marketing resources are bundled for free.
It discuss about the cell, cell theory, Cell Theory Timeline,CELL DIVERSITY- unicellular, multicellular, size of cells, size of cells in human, shape of cell, structure of cell, Microscope, CELL CONSTITUENTS- cell wall/cell membrane, nucleus, cytoplasm in detail
6.Experiments with Xenopus (frog) in the late 1950s demonstrated tha.pdfwailesalekzydelore94
6.Experiments with Xenopus (frog) in the late 1950s demonstrated that cells within the 3 germ
layers had an affinity for each other and thus indicated that they had differentiated enough to be
“biased” toward a specific cell layer (endoderm, mesoderm, ectoderm). Describe an experiment
that demonstrated how the 3 different layers of the gastrula have an affinity for each other.
7.Morphogenesis involves the orchestration of Cell Affinity, Cell Adhesion, and Cell Migration.
How do the adhesive molecules play a role as cells of the embryo change their affinity for one
another?
PLEASE ANSWER BOTH QUESTIONS
Solution
1. Taking an advantage of discovery of amphibian tissues become dissociated into single cells
when placed in alkaline solupans,
2. The prepared single-cell suspensions from each of three germ layers of amphibian embryos
soon after the neural tube had formed.
3. Take two or more of these single-cell suspensions could be combined in various ways. When
the pH of the solution was normalized, the cells adhered to one another, forming aggregates on
agar-coated petri dishes.
4. This experiment shows affinity of 3 different layers of the gastrula.
Cell Affinity: a) The inner surface of the ectoderm has a positive affinity for mesodermal cells
and a negative affinity for the endoderm.
b) While the mesoderm has positive affinities for both ectodermal and endodermal cells.
c) Mimicry of normal embryonic structure by cell aggregates is also seen in the recombination of
epidermis and neural plate cells.
Cell Adhesion: a) cadherins are calcium-dependent adhesion molecules. They are critical for
establishing
and maintaining intercellular connections, and they appear to be crucial to the spatial
segregation of cell types and to the organization of animal form.
b) The cadherins are anchored inside the cell by a complex of proteins called catenins and the
cadherin-catenin complex forms the classic adherens junctions that help hold epithelial cells
together.
Cell Migration: a) migration involves the adhesion of the cell to its extracellular substrate. The
moving cell needs
something to push on, and attaches to the surrounding matrix.
b) Integrins span the cell membrane, connecting the extracellular matrix outside the cell to the
actin cytoskeleton on the inside of the cell. These connections of actin to integrin form focal
adhesions on the cell membrane where the membrane contacts the extracellular matrix.
c) Myosin and its regulators provide the motive force along these actin microfilaments.they are
linked with the lamellipodial actin at the sites of adhesion.
d) Release of adhesions in the rear, allowing the cell to migrate in the forward direction. It is
probable that stretch-sensitive calcium channels are opened and that the released calcium ions
activate proteases that destroy the focal adhesion sites..
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Session Overview
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1. Compression induced formation of
Actin bundle networks in Adherent
Cells
Burhan Saif Addin
Ming Guo, Weitz Lab
Harvard University
ES 298r
Mar 23, 2012
2. Outline
• Introduction and Motivation
• Background Information
• Project Idea
• Results and Discussion
• Conclusion
1
3. 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
4. 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
5. Adherent Cells feel and respond to the
stiffness of their substrate
Esoft~ 1kPa Estiff~ 30-100kPa
Stress versus strain illustrated for different soft tissues
Mechanism for Cellular response to substrate stiffness
are 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.
6. 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
7. Cells on Glass without compression
6
Images are by confocal fluorescence microscopy
9. Compressed Cells on Gel (10% PEG solution)
Volume decrease might be the mechanism that induce actin networks
formation thus cells stiffness.
8
10. Green line Actin’s Dye Fluorescence Intensity across the lines in
is ~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
11. 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. Volker
Schaller et al. 24 June 2010. 10
12. Other method for quantifying actin
bundles networks ?
We attempted to quantify
the 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
13. 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
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