Cellular responses to fluid forces

Pulsed laser-induced tissue ablation: Time-resolved imaging and acute biological effects (Anoop Cherian).

Nanosecond laser surgery with pulse energies in the mJ range is now part of standard clinical practice. Highly focused pulsed lasers (laser microbeams) with energies in the µJ range are also increasingly being used for tissue micro dissection, targeted cell lysis and transfection and cell microsurgery. There is however limited understanding of the physical and biological damage mechanisms of pulsed lasers. Models of cell damage based on experimental data will prove useful in development and refinement of laser based tools for surgery and biotechnology. In the present work we have studied laser induced damage in 3D cell cultures and ex-vivo samples of rat corneas using time-resolved imaging and biological assays. Previously we had shown that time-resolved imaging of laser induced cell lysis could provide great insights into the dynamics of the damage process and also generate quantitative data for calculation of shear forces experienced by cells (see publications). The imaging system we have designed is capable of capturing fast events from nano- to micro-seconds with < 1 m spatial resolution in thick tissue samples. These events have not been studied at such high spatial resolution before and our system can provide a detailed understanding of these processes.

 
 
Biological response of corneal epithelium after laser-induced ablation. Phase-contrast imaging of control (a and c) and ablated (b and d) regions. Confocal fluorescence microscopy of epithelium damage at different depths visualized after actin staining (e-h). Viability of cells at ablation sites visualized en-face (i, j) and in cross-section (k, l) by actin (green) and propidium iodide (red) staining.

 

Laser induced cell transfection and microsurgery (G. Nageswara Rao) 

The use of laser microbeams for cell transfection and microsurgery has been amply demonstrated by several groups. However to date it has not become an established technique mainly due to the numerous parameters that have to be controlled and also due to limited understanding of the damage caused by laser microbeams to cells. We are attempting to standardize this technique for both these applications. Since the group already possesses a detailed understanding of laser-induced damage processes (see above), we can choose the correct parameters for successful transfection or microsurgery but minimize cell death. In the current setup we can deliver 355 or 532 nm, 6 ns focused laser pulses for irradiation of cells or organisms mounted on a motorized stage. Organelles of interest tagged with fluorescent markers can be viewed using epifluorescence, brought to the laser spot and ablated with a few seconds. We plan to upgrade this setup with additional optics to allow simultaneous fluorescence viewing and laser irradiation. Experiments on this setup in collaboration with different groups at NCBS involve transfection of mouse embryonic cells (M. Panickar) and neuronal axotomy in C. elegans (S. Koushika).

 
 
The upper panel shows laser transfection in mouse embryonic cells. In the left-most image a clump of cells is seen prior to irradiation. Three hours after laser irradiation the clump has broken up (middle image). Irradiated cells in the clump show dextran uptake (green) while dead cells marked with propidium iodide (red) (right image). In the lower panel neuronal axotomy in C. elegans is shown. Transport vesicles tagged with GFP are targeted and ablated with < 10 pulses to cut the axon. Arrows point to site of laser focus. (MovieClip)

 

Fluid flow sensing by endothelial cells

Understanding the shear sensing ability of endothelial cells remains a central problem in vascular biology. Based on in-vitro biochemical studies and electron microscopy of blood vessels, it has been proposed recently that the surface layer known as the glycocalyx is the primary sensor of shear effects in endothelial cells. Clinically, it has been observed that the glycocalyx is degraded in conditions of hypoxia, ischemia and atherosclerosis indicating its importance for proper vascular functioning. However, it has been difficult to study the physical properties of the glycocalyx due to its small spatial scale (< 0.2 micron) and its complex composition. Early transmission electron microscopy studies revealed the presence of an electron dense layer that was 50 nm thick on the surface of endothelial cells. This layer termed the glycocalyx was found to be enriched in glycosaminoglycans, proteoglycans, glycoproteins and glycolipids. Our research is focused on understanding the structure and mechanism of fluid flow sensing of the glycocalyx using a variety of microscopy techniques. We have established protocols for isolating human umbilical endothelial vein cells (HUVEC) and maintaining them in culture. We have also fabricated a bench top device for culturing these cells under physiological flow conditions. The device is made from the elastomer, polydimethylsiloxane (PDMS) and is facile and inexpensive to make and is disposable.

 
Bench-top flow chamber for culturing and exposing endothelial cells to fluid flow. (a) Device with persistaltic pump attached in incubator. (b) Close up of the device fabricated in silicone (PDMS). (c) Endothelial cells prior to fluid flow exposure. (d) Same area as (c) post 24 hours flow. Endothelial cells show shape elongation and alignment in the direction of flow.

 

Spatial organization in the endothelial glycocalyx (Amit Sharma and P. Senthil)

We are studying two membrane proteins that form part of the glycocalyx namely syndecan-1 and CD44 to determine their organization within the membrane and their role in fluid flow sensing. We will be conducting hetero-FRET using fragmented antibodies raised against Syndecan-1 and CD44. GFP fusion constructs of syndecan-1 and CD44 are also being used for transient transfection of endothelial cells. These will be used in homo-FRET experiments to determine if these proteins are organized in domains. Transmission electron microscopy of human umbilical vein is also being undertaken to visualize the glycocalyx and identify its components.

Physical properties of syndecan-1 studied by Atomic Force Microscopy (Senthil P. and Lokanath Sai).

We are using biochemical protocols to isolate the heparin sulfate proteogylcan syndecan-1 from endothelial cell surfaces. We wish to study the physical properties of this molecule to understand its role in mechanotransduction. To this end we are attempting to view syndecan-1 molecules using Atomic Force Microscopy. Currently we have standardized protocols for isolating proteoglycans from endothelial cell membranes. Attempts are underway to purify this fraction to isolate intact syndecan-1 molecules. We have also optimized protocols for viewing single molecules of hyaluronic acid, a common extracellular polysaccharide using AFM.

   
Proteoglycan isolation from endothelial cell membranes as seen after SDS-PAGE electrophoresis and silver staining (left). AFM image (right) of the purified fraction shows large numbers of protein clumps.