Mechanics Of Leukocytes
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MECHANICS OF LEUKOCYTES RAJARSHI GUHA (07302009) M.TECH. SEMINAR PRESENTATION GUIDE: Dr. SAMEER JADHAV CHEMICAL ENGINEERING DEPARTMENT IIT BOMBAY
APPLICATIONS OF STUDYING LEUKOCYTE MECHANICS
Leukocytes or white blood cells are the basis of our immune system.
Leukocyte mechanics deals with the force generation and adhesion during recruitment of leukocytes to the inflammation site.
Careful experiments in the laboratory on leukocyte motility mimic what happens in human capillary.
Studying these phenomena can help in making anti-inflammatory drugs.
2
LEUKOCYTE STRUCTURE
There are three major constituent component of a leukocyte: membrane, cytosol and cytoskeleton.
There are two kinds of leukocytes according to their nucleus structure1. PMN leukocytes(neutrophil, eosinophil and besophil) and 2.non PMN(monocyte and lymphocyte).
The cytoskeleton portion is mainly made up of actin. Actin is a globular structural, 42-47 kDa protein.
Individual subunits of actin are known as globular actin (G-actin). G-actin subunits assemble into long filamentous polymers called F-actin.
Two parallel F-actin strands in a helical formation give rise to microfilaments of the cytoskeleton.
3
FUNCTIONS OF CELLULAR COMPONENTS
Membrane maintains a constant, near spherical cell volume.
Membrane mainly produces surface tension, called cortical tension, thus allowing the cell to change its shape during stimulation.
The cytoskeleton is endowed with elastic and viscous properties.
Stimulated cytoskeleton is able to produce active forces that results in spontaneous movement and shape changes.
The cytosol remains mostly passive in terms of force transmission.
4
BIOCHEMICAL ACTIVATION
5
ACTIN POLYMERIZATION AND FORCE GENERATION
There are two kinds of force models widely used to model leukocyte force generation1. network swelling force, 2. membrane polymerization force.
The whole process of polymerization and force generation by network swelling is linked by how the energy of the chemical process of polymerization gets transformed into expansion work.
Polymerizing one additional G-actin monomer at local thermodynamic equilibrium a stress contribution of the order of 6KBT to the network takes place. 6
ACTIN POLYMERIZATION MODELS: BROWNIAN RATCHETS
The physical mechanism is that motion in one direction is allowed by the ratchet, and motion in the opposite direction is prevented by polymerizing monomers.
By adding monomers to its growing tip, a polymer could rectify the free diffusive motions of an object in front of it.
This process produces an axial force transducing free energy of polymerization.
This force makes the random thermal fluctuations of the load or membrane unidirectional.
The BR model has been generalized to include the elasticity of the polymer and to relax the collinear structure of growing tips.
7
ACTIN POLYMERIZATION MODELS: CLAMPED-FILAMENT ELONGATION
It has been observed that motile cells mostly move in 5.4 nm steps and then pauses for a while.
During this time an internal force acts between the filament tethering apparatus (clamp) and the lagging filament.
This can be viewed as "Lock, Load, & Fire" model.
Lock refers to the clamp on the surface containing actin-ATP subunits.
Loading indicates new actin-ADP-Pi addition to the clamp end.
Firing means hydrolysis of ATP by which the clamp translocates and again joins or locks to the filament tip.
8
EXPERIMENT: NEUTROPHIL ASPIRATION
During micropipette aspiration, neutrophil leukocytes exhibit a liquiddrop behavior.
The results show that the outer cortex of the cell maintains a small persistent tension.
The tension creates a threshold pressure below which the cell will not enter the pipette.
A Maxwell-liquid subject to a persistent cortical tension appears to be the most realistic model for entry-flow produced by pipette suction forces.
9
EXPERIMENT: PSEUDOPOD FORMATION
Experiment is done using two pipettes, one holding the neutrophil by gentle suction and the other discharging minute quantities of fMLP.
Cortical tension begins to increase after considerable pseudopod formation and then rises rapidly.
It returns to its baseline value as soon as pseudopod retraction starts.
Pseudopod is devoid of granules which indicate high F-actin cytoskeletal density.
Network polymerization model include a polymerization messenger that is produced at the membrane and diffuses inside the cell with a finite lifetime.
10
EXPERIMENT: CRAWLING INSIDE A MICROPIPETTE
Front edge of a locomoting PMN is a uniformly clear, gray zone devoid of granules which probably is a region of actin gel.
Immediately behind this clear zone is a short section in which many granules undergo rapid, Brownian motions, suggesting that this region is quite fluid.
The rear portion of the cell, containing the nucleus and organelles, shows little internal motion as the cell moves forward.
Progression stops for approximately 16-20 cm H2O counterpressure which is close to human capillary pressure drop.
Velocity varies approximately linearly
11
LEUKOCYTE MOTION: MECHANICAL VISCOELASTIC MODEL
Viscoelastic-solid models are appropriate for modeling the deformation of cells, which possess properties of both fluidity and stiffness.
Fluidity can be modeled by linear viscous dashpots, in which stress is proportional to the rate of strain, with viscosity as the proportionality constant.
Cell stiffness can be represented with linear elastic springs: stress is proportional to strain by a Hookean spring constant.
The cell is divided into six compartments.
The identical inner four compartments consist of a spring, dashpot, and contractile element in parallel; Outer compartments consist of adhesion elements, dashpots and springs in parallel.
12
CONCLUSIONS
These simple models, as actual biological systems are concerned, may not work or fit properly. But they can show control over experiments performed.
Although leukocyte motility is mainly controlled by different molecules and complex chemical signaling, the biophysical treatment gives us lots of insights.
Simulation can be done based on the models with different constraints.
All of the available models don’t describe a leukocyte properly. They sometimes assume leukocyte as a single phase.
They don’t count RBC effects end extra cellular chemical signaling .
Future works should be done considering all these effects.
13
REFERENCES
Dembo, M., W.A. Marganski and Marc Herant. 2003. The Mechanics of Neutrophils: Synthetic Modeling of Three Experiments. Biophys. J. 84:3389-3413
Mogilner, A., and G.Oster. 2003. Force generation by actin polymerization II : The elastic rachet and tethered filaments. Biophys. J. 84:1591-1605
Peskin, C.S., G.M. Odell, G.F. Oster. 1993. Cellular motions and thermal fluctuations: The Brownian ratchets. Biophys J. 65:316-324
Dickinson R.B., and D.L. Purich. 2002. Clamped-filament elongation models for actinbased motors. Biophys. J. 82:605-617
Usami, S., S.L. Wung, B.A. Skierczynski, R. Skalak, and S. Chien. 1992. Locomotion forces generated by a polymorphonuclear leukocyte. Biophys. J. 63:1663–1666.
Evans, E., and A.Yeung.1989. Apparent viscosity and cortical tension of blood granulocytes determined by micropipet aspiration. Biophys. J. 56:151–160.
DiMilla, P.A., K.Barbee, D.A. Lauffenburger. 1991. Mathematical model for the effects of adhesion and mechanics on cell migration speed. Biophys J. 60:15-37.
14
15
APPLICATIONS OF STUDYING LEUKOCYTE MECHANICS
Leukocytes or white blood cells are the basis of our immune system.
Leukocyte mechanics deals with the force generation and adhesion during recruitment of leukocytes to the inflammation site.
Careful experiments in the laboratory on leukocyte motility mimic what happens in human capillary.
Studying these phenomena can help in making anti-inflammatory drugs.
2
LEUKOCYTE STRUCTURE
There are three major constituent component of a leukocyte: membrane, cytosol and cytoskeleton.
There are two kinds of leukocytes according to their nucleus structure1. PMN leukocytes(neutrophil, eosinophil and besophil) and 2.non PMN(monocyte and lymphocyte).
The cytoskeleton portion is mainly made up of actin. Actin is a globular structural, 42-47 kDa protein.
Individual subunits of actin are known as globular actin (G-actin). G-actin subunits assemble into long filamentous polymers called F-actin.
Two parallel F-actin strands in a helical formation give rise to microfilaments of the cytoskeleton.
3
FUNCTIONS OF CELLULAR COMPONENTS
Membrane maintains a constant, near spherical cell volume.
Membrane mainly produces surface tension, called cortical tension, thus allowing the cell to change its shape during stimulation.
The cytoskeleton is endowed with elastic and viscous properties.
Stimulated cytoskeleton is able to produce active forces that results in spontaneous movement and shape changes.
The cytosol remains mostly passive in terms of force transmission.
4
BIOCHEMICAL ACTIVATION
5
ACTIN POLYMERIZATION AND FORCE GENERATION
There are two kinds of force models widely used to model leukocyte force generation1. network swelling force, 2. membrane polymerization force.
The whole process of polymerization and force generation by network swelling is linked by how the energy of the chemical process of polymerization gets transformed into expansion work.
Polymerizing one additional G-actin monomer at local thermodynamic equilibrium a stress contribution of the order of 6KBT to the network takes place. 6
ACTIN POLYMERIZATION MODELS: BROWNIAN RATCHETS
The physical mechanism is that motion in one direction is allowed by the ratchet, and motion in the opposite direction is prevented by polymerizing monomers.
By adding monomers to its growing tip, a polymer could rectify the free diffusive motions of an object in front of it.
This process produces an axial force transducing free energy of polymerization.
This force makes the random thermal fluctuations of the load or membrane unidirectional.
The BR model has been generalized to include the elasticity of the polymer and to relax the collinear structure of growing tips.
7
ACTIN POLYMERIZATION MODELS: CLAMPED-FILAMENT ELONGATION
It has been observed that motile cells mostly move in 5.4 nm steps and then pauses for a while.
During this time an internal force acts between the filament tethering apparatus (clamp) and the lagging filament.
This can be viewed as "Lock, Load, & Fire" model.
Lock refers to the clamp on the surface containing actin-ATP subunits.
Loading indicates new actin-ADP-Pi addition to the clamp end.
Firing means hydrolysis of ATP by which the clamp translocates and again joins or locks to the filament tip.
8
EXPERIMENT: NEUTROPHIL ASPIRATION
During micropipette aspiration, neutrophil leukocytes exhibit a liquiddrop behavior.
The results show that the outer cortex of the cell maintains a small persistent tension.
The tension creates a threshold pressure below which the cell will not enter the pipette.
A Maxwell-liquid subject to a persistent cortical tension appears to be the most realistic model for entry-flow produced by pipette suction forces.
9
EXPERIMENT: PSEUDOPOD FORMATION
Experiment is done using two pipettes, one holding the neutrophil by gentle suction and the other discharging minute quantities of fMLP.
Cortical tension begins to increase after considerable pseudopod formation and then rises rapidly.
It returns to its baseline value as soon as pseudopod retraction starts.
Pseudopod is devoid of granules which indicate high F-actin cytoskeletal density.
Network polymerization model include a polymerization messenger that is produced at the membrane and diffuses inside the cell with a finite lifetime.
10
EXPERIMENT: CRAWLING INSIDE A MICROPIPETTE
Front edge of a locomoting PMN is a uniformly clear, gray zone devoid of granules which probably is a region of actin gel.
Immediately behind this clear zone is a short section in which many granules undergo rapid, Brownian motions, suggesting that this region is quite fluid.
The rear portion of the cell, containing the nucleus and organelles, shows little internal motion as the cell moves forward.
Progression stops for approximately 16-20 cm H2O counterpressure which is close to human capillary pressure drop.
Velocity varies approximately linearly
11
LEUKOCYTE MOTION: MECHANICAL VISCOELASTIC MODEL
Viscoelastic-solid models are appropriate for modeling the deformation of cells, which possess properties of both fluidity and stiffness.
Fluidity can be modeled by linear viscous dashpots, in which stress is proportional to the rate of strain, with viscosity as the proportionality constant.
Cell stiffness can be represented with linear elastic springs: stress is proportional to strain by a Hookean spring constant.
The cell is divided into six compartments.
The identical inner four compartments consist of a spring, dashpot, and contractile element in parallel; Outer compartments consist of adhesion elements, dashpots and springs in parallel.
12
CONCLUSIONS
These simple models, as actual biological systems are concerned, may not work or fit properly. But they can show control over experiments performed.
Although leukocyte motility is mainly controlled by different molecules and complex chemical signaling, the biophysical treatment gives us lots of insights.
Simulation can be done based on the models with different constraints.
All of the available models don’t describe a leukocyte properly. They sometimes assume leukocyte as a single phase.
They don’t count RBC effects end extra cellular chemical signaling .
Future works should be done considering all these effects.
13
REFERENCES
Dembo, M., W.A. Marganski and Marc Herant. 2003. The Mechanics of Neutrophils: Synthetic Modeling of Three Experiments. Biophys. J. 84:3389-3413
Mogilner, A., and G.Oster. 2003. Force generation by actin polymerization II : The elastic rachet and tethered filaments. Biophys. J. 84:1591-1605
Peskin, C.S., G.M. Odell, G.F. Oster. 1993. Cellular motions and thermal fluctuations: The Brownian ratchets. Biophys J. 65:316-324
Dickinson R.B., and D.L. Purich. 2002. Clamped-filament elongation models for actinbased motors. Biophys. J. 82:605-617
Usami, S., S.L. Wung, B.A. Skierczynski, R. Skalak, and S. Chien. 1992. Locomotion forces generated by a polymorphonuclear leukocyte. Biophys. J. 63:1663–1666.
Evans, E., and A.Yeung.1989. Apparent viscosity and cortical tension of blood granulocytes determined by micropipet aspiration. Biophys. J. 56:151–160.
DiMilla, P.A., K.Barbee, D.A. Lauffenburger. 1991. Mathematical model for the effects of adhesion and mechanics on cell migration speed. Biophys J. 60:15-37.
14
15
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