MM2015
Module descriptions
Theory session I (27./28. April)
4 lectures by Gautam Menon (IMSc) and Vijaykumar Krishnamurthy (ICTS)
Topics:
- Random walks, diffusion, polymers, stochastic processes
- Fluid dynamics, Reynold's number, Swimming (as in Purcell's AJP paper), Dipoles and maybe a short introduction to active matter
Theory session II (4./5. May)
2 lectures by Daniel Riveline (University of Strasbourg, France)
Topics: Cell Physics of the Cytoskeleton
- The cell cytoskeleton : polymers, crosslinkers, motors, and the Rho signaling pathway
- Molecular motors in vitro : single motors and collective effects, experiments and theory
- Molecular motors in vivo : single cells and multi-cellular systems, experiments and theory
Project block 1 (27-29. April)
Project: Biochemical manipulations/ traction force microscopy
PI: Aurnab Ghose (IISER Pune)
TA: Sampada Mutalik + Farah Haque
Description: One way to determine the role of molecular players in cell mechanisms is the use of chemical treatments that ideally affect only a specific group of proteins/molecules/reactions in the cell. In this module, we will show the basics how to best do those treatments and how to reduce the risk of artefacts/ non specific effects.
In addition, we will also give an intro to traction force microscopy on single cells using beads embedded in a deformable substrate on which the cell move.
Space: lab bench/ TC CIFF
Microscope: Inverted microscope with fluorescence and DIC/Phase contrast (Mono?)
Requirements: cells (fibroblasts, neuron cells), most of reagents come from Pramod’s lab
Project: Image Analysis
PI: Vijaykumar Krishnamurthy (ICTS)
TA: Chaitra Prabhakara
Description: in order to get from qualitative to quantitative Biology, the ability to extract information and numbers from various kinds of images is an essential tool. Here we will introduce you what kind of information a normal image contains, and methods to gather and interpret this information using programs like Image J and Matlab.
Space: LH3
Requirements: Computers with Matlab, if possible
Project: Cell Stretcher
PI: Bidisha Sinha (IISER Kolkata)
TA: Rinku Kumar + Thanay Bhatt
Description: How to make deformable surfaces that can be used to stretch cells while observing them on the microscope? That was and is a tough challenge, and we will show you in this module the benefits of PDMS and how we can work with it to create surfaces on which cells adhere, even under stress.
We will also show how one can pattern the substrate with regions on which cells do and do not adhere to impose on them a defined geometry.
Space: lab bench/ TC CIFF
Microscope: inverted microscope, fluorescence, Confocal (e.g. LSM 5 live)?
Requirements: adherent cells
Project: AFM on single proteins
PI: ASR Koti (TIFR Mumbai)
TA: Anju Yadav
Description:
Topic 1: Atomic Force Microscope (AFM) based Dynamic Force Spectroscopy (DFS)
In this topic, I will introduce the contact mode operation of atomic force microscope (AFM) for the identification as well as stretching of single-molecules of proteins, DNA and synthetic polymers. Contact mode operation offers a means of applying stretching forces to individual molecules in solution to measure their mechanical response. In this mode of operation, which is known as dynamic force spectroscopy (DFS), the force on the molecule can be kept constant, varied at a constant rate or stretch the molecule at constant velocity, etc. These modes will give information on the viscoelastic, enthalpic and entropic information of the system under tension. Reactions such as protein unfolding, DNA melting (unzipping and shearing), chemical reactions in synthetic polymers can be probed using DFS. In combination with theory and simulations, one can estimate the underlying energy landscape parameters about the energy barrier of activation and potential width from the DFS experiments. All these aspects will be discussed with examples.
Topic 2: Protein unfolding and refolding using dynamic force spectroscopy (DFS) and Cell Adhesion measurements
In this talk, I will introduce protein based DFS experiments to measure protein mechanical stability (force required to unfold). Polyproteins (tandem repeats of proteins that are either chemically crosslinked or produced using molecular biology methods) are typically used in single-molecule pulling experiments. Advantage of polyproteins is that they give a well-defined fingerprint which can be easily differentiated from impurities and also they define the pulling direction. Using DFS, ligand stabilization, protein-protein interactions can be directly measured on single molecules. Recently, is has been used to measure stiffness of proteins as well. Biochemical experiments where protein disulfide bond redox state is modified by enzymes or chemical reducing agents can be probed using DFS. Application of DFS to proteins will be discussed in detail in the talk. I will also talk on cell-receptor and cell-cell interactions that can be probed using DFS and the type of information that these experiments could give to understand cell mechanics both qualitatively and quantitatively.
Space: lab bench
Microscope: AFM
Requirements: AFM tips, Mica
Project: Supported lipid bilayers
PI: Thomas Pucadyil (IISER Pune)
TA: Sukrut Kamerkar
I would instead like to discuss a new model membrane system of supported membrane tubes which we find are far superior to conventional models in terms of reaching a mechanistic understanding of membrane fission...and its also breathtaking when one see dynamin acting to cut these tubes. Attached is our recent paper describing the use of this system to reconstitute epsin-mediated clathrin polymerization.
Space: lab bench
Microscope: Inverted fluorescence microscope with time lapse imaging capability (Mono or alternative)
Requirements: Thomas brings everything
Project block 2 (30.4. – 2.5.)
Project: Optical Tweezers
PI: Roop Mallik (TIFR Mumbai)
TA: Divya Pathak + Joseph Thottacherry + Susav Pradhan
Description: We use optical tweezers to measure the force that motor proteins exert in order to carry cargo such as phagosomes and lipid droplets along microtubules. These experiments are done inside cells as well as using in vitro assays where we use phagosomes and lipid droplets purified from cells. Our goal is to understand why various motors are designed differently at the single-molecule level, and how these differences allow these motors to work together in a team.
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Calibration of optical trap for beads of known sizes
Trapping of E Coli cells and assessment of Photodamage
Trapping of phagosomes inside dictyostelium cells (not sure if this will be possible, but we can try)
Space: lab bench
Microscope: Zeiss Axiovert 200 (with Thorlabs OT)
Requirements: maybe Dictyostelium, Pondwater
Project: Force probe
PI: Pramod Pullarkat (RRI)
TA: Seshagiri Rao + Lama Prakash
Description: The force probe apparatus is another tool to apply or measure forces on cells, especially for elongated cells like neurons or myocytes. You will learn how to install and operate it.
Space: lab bench, TC Ciff
Microscope: inverted microscope (LSM 5 live)
Requirements: Pramod brings everything, maybe trip to RRI for demonstration
Project: AFM on proteins and cells
PI: Gautam Soni (RRI)
TA: Sourabha
Description: AFM as a tool to pull and push cells. We show how to do it!
Space: lab bench
Microscope: AFM (same as Koti)
Project: Supported lipid bilayers
PI: Thomas Pucadyil (IISER Pune)
TA: Sukrut Kamerkar
Description:
I would instead like to discuss a new model membrane system of supported membrane tubes which we find are far superior to conventional models in terms of reaching a mechanistic understanding of membrane fission...and its also breathtaking when one see dynamin acting to cut these tubes. Attached is our recent paper describing the use of this system to reconstitute epsin-mediated clathrin polymerization.
Space: lab bench
Microscope: Inverted fluorescence microscope with time lapse imaging capability (Mono or alternative)
Requirements: Thomas brings everything
Project block 3 (3.-5.5.)
Project: Tissue stretcher
PI: Namrata Gundiah
TA: Lama Prakash
Description: We will show how to extract the stress-strain relationships in substrates which are stretched in two directions simultaneously. The substrates are PDMS (isotropic) or may also include tissues with directional layouts (anisotropic) which will have different responses in the two directions. Such methods are especially important in testing growth and remodeling hypothesis related to cells cultured on substrates and exposed to varying biaxial stretches/ stresses.
Space: lab bench
Custom biaxial testing instrument, CCD camera for video extensometry
Project: Micropipette aspiration
PI: Darius Köster (NCBS)
TA : Chaitra Prabhakara
Micropipette aspiration is a versatile and easily to install tool that can be used to apply a controlled suction force to cells in order to measure the amount of excess membrane or to measure their elasticity. We will install a simple version and see how to calibrate it as well as how cells react to suction.
Space: Microscope
Microscope: LSM 5live
Requirements: pulled glass needles, cells in suspension
Project: Force probe
PI: Pramod Pullarkat (RRI)
TA: Seshagiri Rao + Lama Prakash
Description: The force probe apparatus is another tool to apply or measure forces on cells, especially for elongated cells like neurons or myocytes. You will learn how to install and operate it.
Space: lab bench, TC Ciff
Microscope: inverted microscope (LSM 5 live)
Requirements: Pramod brings everything, maybe trip to RRI for demonstration
Project: Patterned and deformable substrates
PI: Feroz Menon (C-Camp)
TA: Chaitra Prabhakara
Description: How to make deformable surfaces that can be used to stretch cells while observing them on the microscope? That was and is a tough challenge, and we will show you in this module the benefits of PDMS and how we can work with it to create surfaces on which cells adhere, even under stress.
We will also show how one can pattern the substrate with regions on which cells do and do not adhere to impose on them a defined geometry.
Space: lab bench/ TC CIFF
Microscope: inverted microscope, fluorescence, Confocal (e.g. LSM 5 live)?
Requirements: adherent cells
Eventually one more image analysis module?