Cytomechanics 432/532

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Cytomechanics432/532

• Tuesday, January 18, 2005 Introduction
• WebCT syllabus, book, resources, posting.
• Office BME 124 Weds, Thurs 1-4 PM
• Grading HW Exams Project
• Craelius_at_rci

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Learning objectives

• 1. To learn the structural/mechanical components
  of cells, specifically biophysics and material
  properties of the cytoskeleton (CSK), membrane,
  and matrix.
• 2. To learn about experimental tools for
  evaluating cell mechanical properties,
  specifically mechanical testing, imaging with
  immunocytochemisty and knock-out methods.

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Learning Objectives

• 3. To learn kinematics and dynamics of cells,
  specifically, interactions among CSK, cytosol,
  matrix, and nucleus, mechanotransduction, and
  motility
• 4. To learn statistical mechanics of cell
  polymers and CSK assembly.
• 5. To learn tools for modelling cell mechanics,
  specifically simulations with matlab and
  simulink.

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Topics in Cytomechanics

• A cell is a cytoplasmic structural element.
• Tensegrity holds it together -centripetally.
• Structural components include lipids, and 3
  separate filament systems.
• No cell is an island- interactions with others
  and the ECM shape and regulate it.
• Trans-skeletal molecules regulate the cell.
• Rxns in solid-state versus enzyme solution.

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Questions

• How do cells
• maintain and change shape?
• Move?
• Grow and maintain a size?
• Anchor to substrate or stick together or not?
• Transport materials inside?
• Form tissues?
• Sense force and deformation?

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Applications of Cytomechanics?

• Medical
• Stress-Growth Hypothesis
• Mechanoelectrical Feedback
• Tumor-Endothelium
• Wound Healing
• Edema
• Bone Cartilage Control
• Cellular signalling

• Technological
• Gas structural elements
• Motility of Gels
• Microtubular nanostructures
• Bioprocess optimization
• Plant Growth Production
• Microgravity Effects

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How are cells put together?
Not nice and regular Varied and irregular
200 different types
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The generic cell
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Tension compression hold the cell together
Green fluorescent dye for Actin
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Basic Cell Components

• A membrane, skeleton, and internal structures.
• All serve both as structural and functional
  elements.
• Simplified basic building blocks

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Geodesic- Buckminster Fuller
A geodesic dome uses a pattern of self-bracing
triangles in a pattern that gives maximum
structural advantage, thus theoretically using
the least material possible. (A "geodesic" line
on a sphere is the shortest distance between any
two points.)
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Stick Geodesic Domes Ingber
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Tensegrity structures

• Body stands upright by compression due to gravity
  counteracted by tension from muscles
• Same for bridges and many other structures.

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100 nM
Tensegrity
Neurofilaments Cross-linked In frog axon
Spectrin In RBC
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the CSK smart design

• orienting along stress lines, filaments size
  themselves according to strength requirements a
  conservative architectural practice.

Thin supporting struts connecting thick beams
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Underneath the hood

• Lipid shell
• Actin network
• Cytosol
• Filaments
• Organelles
• Nucleus

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Lipid vesicles are ghost-like

• pipets suck up the vesicles
• Miscibility allows intermingling

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Plasma membrane

• Lipid bilayer 30 A
• Dielectric - capacitor
• Amphiphile
• Semi-permeable
• No tensile but some shear strength

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The cytoskeleton
Decorated actin
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Tensegrity
Malines, Belgium
Fibroblast
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Major Filaments

• Filaments
• Actin 8 nM
• Intermediate 10nM
• Microtubules25 nM

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3 types of filaments
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Cellular Rods and Ropes
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Filaments have different functions
Spectrin bends
Microtubules are stiff
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Cell Crawling
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Visualizing actin-myosin motion
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Types of motors
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How does the CSK provide structure?
Signals travel at speed of sound.
Some results are not compatible with tensegrity
model
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Structure by light immunofluorescence
PMT
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Fibroblasts are stained with Phallacidin green
for F actin, Texas red for microtubules, and DAPI
for nucleic acid.
F actin
microtubules
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F actin is green with Phalloidin, G actin is
red with Texas red. Nucleus has fewer stress
fibers, but is thicker than rest of cell, so red
is diffuse.
F actin
G actin
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Fibroblast dividing
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Cells are Wiggly and Soft
New ways to describe softness- difference between
cooked and uncooked noodles. thermal
fluctuations Of lipid vesicle
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Types of Loading
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Swelling and Lysis to measure membrane strength

• RBCs

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Muscle
Frog
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Pipet Aspiration
Neutrophils are WBCs involved in immune
response. The source of cortical tension is
unknown, but may be from actin tangential to
surface.
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Unwinding of rubber
Rubber Elasticity
s
1 mm
e
Collagen
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Stress-strain varieties
liquid
s
J Curve
Rubber
unwinding
e
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Solutes
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Elasticity and safety at high strains Mesangial
cell area expansivity
Rubber-like
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Common Quantities in Cytomechanics
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Where we are going

• Feedback Regulation Bioelectricity- eg. Heart,
  bone, cartilage
• Optics of cytoskeleton immunofluoresc.
• Micromotors Gels piezo- ferro-electric
• Cell shape regulation, eg. Edema, tumors
• Tissue morphogenesis osseointegration
• Endothelial regulation
• Wound healing

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Common quantities
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The cytoskeleton is both internal and external
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Fibroblast-myocyte interactions
Fibroblasts
Myocytes
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Growth patterns vary in myocytes
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Wall stress in a thick sphere

• To find equilibrium forces
• S Fup SFdown

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Membrane Tension
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• Cells will adhere to specific islands, properly
  coated.
• The traction force can be seen by the bending of
  the substrate.
• Microfabricated culture wells allow cell to make
  many E connections.

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Shape Determined by Stress
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Knock-out methods

• Spectrin
• Actin
• Microtubules
• Intermediate Filaments

• Heat
• Cytochalasin
• Nocodazole
• Acyrlamide

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Pulling Chromosomes out
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Cartilage
pKapH logHA A-
Polymer charge determines Swelling
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Tensegrity Industry
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Designer foam
Zero Mean curvature
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Percolation theory of the CSK assembly

• Rule network evolves by random connections
  between 2 active sites, each with some site
  occupation probability, p. A cluster is a set of
  occupied sites all of which are connected either
  horizontally or vertically, i.e. an occupied site
  belongs to a cluster if a member of the cluster
  is either above, below, left, or right of it. A
  spanning cluster has an element in both the top
  and bottom rows of the site matrix.

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Proteins

• Primary, secondary, tertiary, quaternary
  structures
• Make filaments rods, tubes,
• Flexural Stiffness

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Polymer bendingassuming it is a thin rod
bend
L
R
q
?
At finite temperature, an otherwise straight rod
bends as it exchanges energy with its
environment. It bends more as T rises, like a
noodle.
Landau Lifshitz, Theory of Elasticity, 1986.
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What persistence length means
When L ep , what happens at Earc kT?
Thus a rod of the persistence length is curved
at 81 degrees when the thermal energy scale
reaches kT. If L ltlt ep then rod is relatively
straight, otherwise not. Persistence length sets
the scale of thermal fluctuations. Filaments
with countour length gtgt ep are highly convoluted
and can assume many configurations.
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Entropic springs
4-segment chain configurations
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tension
Small ree Many Config- urations
Large ree Few Configurations

Applying a tension to the zero ree state reduces
possible configurations to 10. S drops from
ln(16) to ln (10). Hence tension translates to
loss of entropy.
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Effective spring constant for a convoluted chain
near equilibrium
Have you seen a model of this?
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Tissue

• Aggregate cells are more complicated. Many
  different types of connections, each with their
  own biochemical traffic patterns

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Methods of cell regulation Signalling by
mechanotransducers current Molecular CSK
regulators Integrin Nuclear transcription
protein
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Exercise

• Prove that a hollow design is advantageous for a
  microtubule. Assume an outer and inner radii of
  14 and 11.5 nM, respectively, and compare this
  with a solid MT of the same outer radius. What is
  the most efficient way for proteins to gain
  rigidity, ie. on a per unit mass basis?

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• Review Questions
•  The cytoskeleton is made of (Select one)
• Filamentous protein
• Lipid
• Actin, microtubules and microfilaments
• Extracellular matrix (ECM)
• (a, b and c)
• (a and c)
• (all of the above)
• Integrin is a transmembrane peptide (True or
  False)
• State a specific method or technique to "knock
  out" or remove a component of the cytoskeleton
•  
• Immunofluorescence is a procedure to visualize
  specific molecules in a cell. The technique
  involves shining long wavelength light on the
  specimen, and seeing or detecting shorter
  wavelength light fluoresce. (True or false).