Microfluidic Manipulation and Measurement on Blood Cells: From Synthetic Micro-thrombosis to in vitro Pressure Measurements Relevant to the Microcirculation.   Understanding the flow of blood cells in small capillaries is a constant scientific challenge to further investigate certain aspects of microcirculatory diseases like sickle cell and hemolytic anemia or thrombosis. The flexibility, as well as the small-scale nature of the flow that microfluidic approaches offer, make it the perfect and ubiquitous candidate to explore properties of blood flow at micronscale. Through two examples, I will show how microfluidic techniques allow to revisit an old physiological phenomenon (the so-called Fahraeus effect) and how to make a difficult pressure measurement come true at the single cell level.  Contributors :  Manouk Abkarian, Magalie Faivre, Howard A. Stone

Single Molecule Studies of Protein Function.  Direct visualization of the conformational dynamics of proteins and nucleic acids using fluorescence microscopy techniques.  Also, studies of the photophysics of Cy5, a fluorescent marker in widespread use.

Long Term Thermal Memory and Learning in C. elegans.  The nematode C. elegans establishes a long-term thermal memory by associating temperature with the presence of food. Several mutations cause defects in thermal learning, possibly shedding light on the molecular details of this process. Ongoing experiments are aimed at further analysis of the roles of various signaling molecules in the formation, storage and retrieval of thermal memory.

Nanopore Technology.  A novel technology for probing, and eventually sequencing, individual DNA molecules using single-channel recording techniques has been conceived. Single molecules of DNA are drawn through a small channel or nanopore that functions as a sensitive detector. The detection schemes being developed will transduce the different chemical and physical properties of each base into a characteristic electronic signal. Nanopore sequencing has the potential of reading very long stretches of DNA at rates exceeding 1 base per millisecond.  See also:  http://www.mcb.harvard.edu/Faculty/Branton.html and  http://mcb.harvard.edu/branton/index.htm

Unbinding and Unzipping With Magnetic Tweezers.  Magnetic tweezers allow massively parallel measurements on many individual single molecules being separated at a constant force. The separation of double stranded DNA into single stranded DNA known as unzipping was studied at constant force and constant temperature for temperatures between 15°C and 50°C and compared with theoretical models. A similar experiment using magnetic tweezers has been done to probe the dissociation of ligand-receptor complexes such as biotin-streptavidin (avidin) and sulfonamide-carbonic anhidrase.

Is the Flagellar Motor of E. coli a Viscometer? (i.e., Can It Sense Load?)  The amount of torque produced by the motor profoundly affects its switching behavior.  My goals are to 1) understand the mechanistic links involved; 2) characterize other behaviors influenced by switching; 3) reveal whether load information is discerned usefully by the cell (i.e., what is the nature of feedback, if any).

"Barotaxis" Quantitative Measurement of C. elegans Response to Pressure.  C. elegans respond to changes in hydrostatic pressure. We have measured this response using specific pressure stimuli, revealing a linear modulation of the worm’s reorientation frequency. Using genetic analysis and laser ablation studies we are working to identify the neurons and neural signaling involved in pressure sensation as well as the worm's time-integrated response.

Modeling Planar Flagellar Motions

Mechano-sensitive membrane proteins.  We study the relationship between lipid bilayer properties and the structure and dynamics of a transmembrane protein.  Specifically, we’re studying how the two transmembrane helices of CD39, a membrane bound nucleotidase with an extracellular active site, interact, regulate activity, and respond to changes in the mechanical properties of the lipid bilayer.  We look at the dynamic as well as the structural features of the transmembrane helices as a means to understand the interplay among bilayer, transmembrane helices, and enzymatic active site.

Mechanical properties of organized whole chromosomes.  I am development methods for examining the axial flexibility of organized meiotic chromosomes and for studying the basis for chromosome motion in living cells.

Dynamics of RNA Translocation.  The dynamics of RNA translocation are studied theoretically using a free-energy landscape picture.  It is shown through computer simulation of RNA folding that random RNAs behave like a string of simple hairpins when translocated.  This work had relevance to many biological processes such as ribozomal protein synthesis.

Subcellular Nanosurgery Using Femtosecond Laser Pulses.  We performed laser nanosurgery in live cells, where we ablated a single mitochondria and severed cytoskeletal filaments without compromising the cell membrane or the cell’s viability. We also cut dendrites in living C. elegans without affecting the neighboring neurons. This dissection technique enables non-invasive manipulation of the structural machinery of cells and tissues down to several-hundred-nanometer resolution.

Cellular Organelle Imaging and Chemical Analysis via FIB.  Data on the evolution of cells is sought via a comparative FIB imaging/chemical analysis of Archaea, Eubacteria and Eukaria with particular reference to those organelles harboring ether-linked lipids.

Folding Dynamics of Individual Ribonucleoproteins.   Complexes of RNA and protein, ribonucleoproteins such as the ribosome or spliceosome, play many roles in the molecular biology of the cell. We are studying a minimal one-protein, one-RNA system, the bI5 ribozyme, using single-molecule FRET techniques. We observe folding and splicing of the ribozyme, a group I intron which catalyzes its own excision. We observe a new weak binding mode of the protein, CBP2, to the bI5 RNA, and multiple pathways for folding. We conclude that the protein binds rapidly to the RNA and induces folding to the native state.

Spore Discharge in Mushrooms: The Surface Tension Catapult.  We are interested in the active mechanism of spore ejection in some fungi based on the coalescence of a micron-size water droplet onto the spore. Most of the common edible mushrooms use this mechanism to discharge their spores into the air at an initial speed of approximately 1 m/s. We used an ultra-high-speed camera to study this phenomenon.  Our detailed observations and measurements confirm one of the theories proposed for the discharge mechanism, we have also developed a more detailed model allowing a better understanding of the process.

Live Cell Single-particle Tracking Reveals a Novel Endocytic Pathway.  Live cell two-color fluorescence imaging has revealed a novel endocytic pathway initiated by the binding of cationic molecules to anionic proteoglycans on the cell surface.  This pathway is dominated by the direct entry of cargo into late endosomes independently of early endosomes or microtubule-dependent transport.  Current research is aimed at determining the transport mechanism responsible for the direct entry of cargo into late endosomes.

Mechanisms of Peptide Selection and Translocation by the Transporter associated with Antigen Processing (TAP).  TAP uses ATP to pump peptides into the endoplasmic reticulum where they are loaded onto MHC molecules for immune surveillance. Our aim is to understand the mechanism of transport, particularly how ATP hydrolysis is coupled to conformational changes, and how specific peptide sequences are selected through determining a high-resolution structure of TAP.

Cell Motion and Mechanics in Glioblastoma Multicellular Tumor Spheroids.  We seed glioblastoma multicellular tumor spheroids into a collagen matrix and observe the migration patterns of cells invading the gel away from the tumor core. We wish to relate the migratory behavior of cells with the local mechanical environment of the cells, e.g. collagen fiber traction, destruction and digestion.

Probing the Nucleus.  The mechanical properties of the nuclear lamina for both wildtype and lamin A/C deficient nuclei were estimated by conducting sharp tip probe indentation on isolated nuclei and interpreting the experimental results using a novel computational model. The effect of lamin A/C deficiency on the nuclear structure was studied by comparing the material properties of Lmna-/- nuclei to those of Lmna+/+, providing some insight for better understanding of the etiology of laminopathies promoted by lamin mutations.

HEAT Repeat Domains as Protein Springs.  Many molecules can act as springs with the function of detecting mechanical stress exerted by the expansion/contraction of their substrates. We are characterizing PIKKs as possible molecular springs: the structural unit is the HEAT domain, two short, interacting, antiparallel helices organized in flexible higher order gyres composed of 40-60 domains. We are exploring both single molecule and ensemble methods for measuring force extension curves related to elastic properties of individual proteins.

Plant hybraulics.  My research project is to understand aspects of plant hydraulic systems, design features of the transport path and plants’ ability to control and distribute fluxes within the organism.