MBL Physiology Course

I participated in the Physiology Course at the Marine Biological Laboratory during the Summer of 2008. The Physiology Course is a 7-week long cell biology "boot camp". There are lectures and seminars every day given by biologists from around the world. The first week is instructional, and they teach you basic biochemical techniques, microscopy, image analysis, coding and more. The following 6 weeks are divided into 3 research sessions, during which you work with professors on a project they've prepared for the course. Overall, it was a great experience and I learned a ton about how cell biologists think about and approach questions. It was also astounding how many fundamental questions in cell biology still haven't been answered. I would also like to sincerely thank the Scholars of The Bauer Center for Genomics Research at Harvard for their financial support.

Here are the three projects I worked on in the research sessions:

Session I - Actin density and lamellipodium thickness
Dan Fletcher & Julie Theriot
How does actin density vary in the cell lamellipodium? It is believed that actin density is greater at the leading edge of migrating cells than in the rest of the lamellipodium. This has implications for the dynamics of actin assembly during cell protrusion and migration. Previous measurements of cell thickness have been performed primarily on fixed cells, however it is unknown if the fixation process introduces thickness artifacts. In order to determine actin density in live cells, we: Performed AFM measurements on live and adherent Drosophila S2 cells, live motile keratocyes, and fixed keratocytes Measured the fluorescence microscopy of phalloidin-stained F-actin in fixed cells Correlated the thickness measurements and phalloidin intensity to calculate an average distribution of actin density	AFM error image of the top-left of a live S2 cell.
Further Reading Abraham VC, Krishnamurthi V, Taylor DL, Lanni F. "The actin-based nanomachine at the leading edge of migrating cells." Biophys J. 1999 Sep;77(3):1721-32. Laurent VM, Kasas S, Yersin A, Schäffer TE, Catsicas S, Dietler G, Verkhovsky AB, Meister JJ. "Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy." Biophys J. 2005 Jul;89(1):667-75. Iwasa JH, Mullins RD. "Spatial and temporal relationships between actin-filament nucleation, capping, and disassembly." Curr Biol. 2007 Mar 6;17(5):395-406.

Session II - Mechanisms of centrosome assembly
Tony Hyman & Frank Jülicher
How do centrioles nucleate centrosome assembly and what is the mechanism of the assembly process? The centrosome, composed of centrioles surronded by a pericentriolar matrix (PCM) of proteins, is the microtubule organizing center of the cell, and is critical for mitosis. Several proteins are known to be comprise the PCM (e.g. SPD-2, SPD-5), however the nucleation and assembly mechanisms are unclear. To address these uncertainties, we: Performed time-lapse, spinning-disk confocal microscopy of centrosome assembly by looking at GFP-fused PCM proteins in C. elegans embryos Used fluorescence recovery after photobleaching (FRAP) to measure the movement and recovery of various PCM proteins Developed a theoretical model for the mechanism of PCM growth and simulated it computationaly	MATLAB simulation of PCM assembly around the centrioles.
Further Reading Kirkham M, Müller-Reichert T, Oegema K, Grill S, Hyman AA. "SAS-4 is a C. elegans centriolar protein that controls centrosome size." Cell. 2003 Feb 21;112(4):575-87. Pelletier L, Ozlü N, Hannak E, Cowan C, Habermann B, Ruer M, Müller-Reichert T, Hyman AA. "The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication." Curr Biol. 2004 May 25;14(10):863-73. Hamill DR, Severson AF, Carter JC, Bowerman B. "Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains." Dev Cell. 2002 Nov;3(5):673-84.

Session III - A ParM ortholog encoded by the plasmid pb171
Dyche Mullins
How do low-copy number plasmids segregate themselves across the cell such that they are inherited upon cell division? The E. coli R1 plasmid contains a par locus of sequences that encode the protein machinery that segregates the plasmids before cell division. Particularly, ParM is an actin-like protein that assembles into filaments that bind to and move the plasmid. ParM is quite an unique protein, as it exhibits dynamic instability similarly to microtubules. Whereas the Par proteins of the plasmid R1 have been well characterized, the plasmid pb171 has been studied to a much lesser extent. The pb171 equivalent of ParM has only ~40% sequence identity to R1 ParM, and we are thus curious as to whether it functions similarly to R1 ParM. To study these plasmid segregation systems, we: Used FRET to study the structure of pb171 ParM polymers Used TIRF microscopy to observe dynamic instability in R1 ParM, and look for dynamic instability in pb171 ParM Performed light scattering measurements of the assembly kinetics of both R1 and pb171 ParM's Reconstituted the R1 segregation system in vitro using DNA-coated beads, and reconstituted the same system with pb171 ParM	In vitro reconstituted R1 plasmid segregation system from purified proteins.
Further Reading Garner EC, Campbell CS, Mullins RD. "Dynamic instability in a DNA-segregating prokaryotic actin homolog." Science. 2004 Nov 5;306(5698):1021-5. Garner EC, Campbell CS, Weibel DB, Mullins RD. "Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog." Science. 2007 Mar 2;315(5816):1270-4. Orlova A, Garner EC, Galkin VE, Heuser J, Mullins RD, Egelman EH. "The structure of bacterial ParM filaments." Nat Struct Mol Biol. 2007 Oct;14(10):921-6. Ebersbach G, Gerdes K. "The double par locus of virulence factor pB171: DNA segregation is correlated with oscillation of ParA." Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15078-83.