The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F- actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus

Three-dimensional image cytometer based on widefield structured light microscopy and high-speed remote depth scanning

A high throughput 3D image cytometer have been developed that improves imaging speed by an order of magnitude over current technologies. This imaging speed improvement was realized by combining several key components. First, a depth-resolved image can be rapidly generated using a structured light reconstruction algorithm that requires only two wide field images, one with uniform illumination and the other with structured illumination. Second, depth scanning is implemented using the high speed remote depth scanning. Finally, the large field of view, high NA objective lens and the high pixelation, high frame rate sCMOS camera enable high resolution, high sensitivity imaging of a large cell population. This system can image at 800 cell/sec in 3D at submicron resolution corresponding to imaging 1 million cells in 20 min. The statistical accuracy of this instrument is verified by quantitatively measuring rare cell populations with ratio ranging from 1:1 to 1:10[superscript 5].National Institutes of Health (U.S.) (Grant 9P41EB015871-26A1)National Institutes of Health (U.S.) (Grant 5R01EY017656-02)National Institutes of Health (U.S.) (Grant 5R01 NS051320)National Institutes of Health (U.S.) (Grant 4R44EB012415-02)National Science Foundation (U.S.) (Grant CBET-0939511)Singapore-MIT Alliance for Research and TechnologyMIT Skoltech InitiativeHamamatsu CorporationDavid H. Koch Institute for Integrative Cancer Research at MIT (Bridge Project Initiative

Genosenor Technology Development

Contains table of contents for Part IV, table of contents for Section 1, and reports on two research projects.Genometrix, Inc. Contract GMX-GH00776-04Defense Advanced Research Projects AgencyU.S. Air Force - Office of Scientific Researc

Sub-population analysis based on temporal features of high content images

Background: High content screening techniques are increasingly used to understand the regulation and progression of cell motility. The demand of new platforms, coupled with availability of terabytes of data has challenged the traditional technique of identifying cell populations by manual methods and resulted in development of high-dimensional analytical methods. Results: In this paper, we present sub-populations analysis of cells at the tissue level by using dynamic features of the cells. We used active contour without edges for segmentation of cells, which preserves the cell morphology, and autoregressive modeling to model cell trajectories. The sub-populations were obtained by clustering static, dynamic and a combination of both features. We were able to identify three unique sub-populations in combined clustering. Conclusion: We report a novel method to identify sub-populations using kinetic features and demonstrate that these features improve sub-population analysis at the tissue level. These advances will facilitate the application of high content screening data analysis to new and complex biological problems.Computation and Systems Biology Programme of Singapore--Massachusetts Institute of Technology Allianc

Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.Massachusetts Institute of Technology. Presidential FellowshipNational Institutes of Health (U.S.). Biotechnology Training FellowshipSingapore-MIT Alliance for Research and TechnologyMassachusetts Institute of Biotechnology Training GrantMassachusetts Institute of Technology (Open-source Funding

Wireless-compatible CMOS-based celluar sensor

The title as published reads "celluar sensor" but should be "cellular sensor."Biosensors have a need to accurately detect and quantify a biological target within a complex medium, such as serum. Cells are uniquely qualified to accomplish this, but cannot communicate with the outside world. We demonstrate a synergistic fusion between a genetically engineered cell line and a wireless-compatible CMOS sensor leveraging the cells innate ability to specifically bind, amplify and transduce the signal with electronics that are capable transforming this cellular signal into an electronic one. We demonstrate the detection of TNF-alpha (TNFalpha) within serum without washing. Since cells are real-time transducers of binding events, real-time monitoring of biological agents and reactions are possible

Partial reconstruction of micro,villus core bundle: characterization of villin as a Ca++-dependent, actin-bundiing/depotymerizing protein

ABSTRACT The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass NO polypeptide, villin. This report describes the purification and characterization of villin as a Ca"-dependent, actin bundling/depolymerizing protein. The 100,000 g supernatant from a Ca " extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M r (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. The viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca " concentration in solution. At low free Ca " (<10-6 M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca " (>10-6 M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer