David A. Knecht 
Principal Investigator

Professor of Molecular and Cell Biology
Director, Light Microscopy Facility

Department of Molecular and Cell Biology
University of Connecticut 
BPB 307
91 North Eagleville Rd
Storrs, CT 06269
david.knecht@uconn.edu



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Actin Binding Proteins in Cell Dynamics and Disease:
 

Regulation of Actin Cytoskeletal Architecture by Fimbrin A
The fimbrins, also known as plastins, are members of the Calponin Homology superfamily of actin-binding proteins. Fimbrin has two calcium-binding domains (EF hands) and has been shown to have calcium regulated actin-binding activity in vitro in some organisms. This protein is unique among the CH domain containing proteins in possessing a tandem repeat of the actin-binding domain (ABD) within a single polypeptide chain. In order to understand what regulates fimbrin’s binding to actin filaments in the cell, we generated fluorescent fusion proteins, encoding either the entire protein or its various domains, and examined the localization of these fluorescent proteins in living cells. Fimbrin localizes to newly formed pseudopods as well as macropinosome cups, but only weakly in the peripheral cortex. Deletion of the EF hands (∆EF) had no discernable effect on protein localization However, the first actin-binding domain (ABD1) alone or the EF hands plus the first actin-binding domain (EA1) both localized more strongly to the cortex of the cell than the whole protein. Surprisingly, expression of the second actin-binding domain (ABD2) alone leads to the formation of a large aggregate of actin filaments. The actin aggregates induce by ABD2 were further investigated using the tetracysteine tagging system. We found that ABD2-4cys was still able to generate actin aggregates, indicating that protein association through the GFP domain did not lead to filament aggregation. We have also used bacterially expressed fimbrin and its various domains to further investigate the function of the two actin- binding domains of fimbrin in cell free systems. The two actin-binding domains of fimbrin have different affinities for pure F-actin and induce different architectures of F-actin networks and have different effects on filament stability. These results suggest that the two ABDs are functionally different and cooperate in defining the function of the whole proteinRan-der1.htmlshapeimage_6_link_0

Role of T-plastin in regulating actin network architecture and reorganization during cell migration

The first step in cell motility is the forward protrusion of the cell membrane due to the remodeling of the underlying actin network. Actin cross-linking/bundling proteins are important mediators of the formation of actin network architectures, including lamellipodia and filopodia. There are a large number of actin binding proteins and it is currently unclear the extent to which they serve unique versus overlapping functions. Here we report a specific novel function for the actin cross-linking/bundling protein T-plastin (Pls3). T-plastin normally localizes to the lamellipodium and transiently to stress fibers and focal adhesions. Depletion of T-plastin results in the collapse of the lamellipodium, leaving behind a dendritic network of F-actin filaments.  The residual filaments can grow bi-directionally, either toward the lamellipodium in dendritic form or linearly toward the cell body. T-plastin deficient cells also display periodic stress fiber-like actin structures in the lamella. We conclude that T-plastin is an important actin binding protein that is necessary for the proper organization of the actin network at the leading edge during cell migration. Fei1.htmlFei1.htmlshapeimage_7_link_0shapeimage_7_link_1
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Macropinocytosis
Macropinocytosis is a major endocytic pathway in living organisms characterized by non-selective, clathrin-independent and receptor-independent bulk-phase uptake of extracellular fluid. The process proceeds by the formation of actin-driven protrusions of the plasma membrane creating cup-like structures that fuse distally to form vesicles ranging from 0.6 to 5 um in diameter. Macropinocytosis is important for many biological processes including nutrient uptake, membrane recycling, antigen processing and pathogen invasion however, much about its spatiotemporal regulation still remains unstudied. Our current study utilizes Dictyostelium discoideum, a genetically tractable organism, as a model system to identify new genes that regulate macropinocytosis.We have used REMI mutagenesis and flow cytometry to screen for cells defective in macropinocytosis, and using the inverse PCR technique, we were able to isolate two genes that were disrupted in some of our mutants. We are currently focused on phenotype characterization of the mutants, recapitulation of REMI mutation, rescue of the disrupted gene and construction of GFP fusion probes to visualize the dynamics of  ‘agaA’ and cysA localization during macropinocytosis by live cell imaging.Charito1.htmlshapeimage_17_link_0
Silica-induced Cell Death in Macrophages 

Occupational exposure to crystalline silica is a serious health hazard. Manifestation of this disease depends on intensity and duration of exposure to silica. All work done until now has dealt with population analysis of cells. We are examining the role of caspases and intracellular organelles in cells exposed to silica at the single cell level. Using Fluorescence Resonance Energy Transfer (FRET) probes directed towards caspases and Bid along with mitochondrial marker Tetra Methyl Rhodamine Ester (TMRE) it has become possible to study interactions between mitochondria and these proteins. Alterations in the mitochondrial membrane potential vary in cells undergoing apoptosis and necrosis. Changes in lysosomal permeability, nuclear morphology, and phosphotadityl serine (PS) externalization have been examined with their respective probes. Reactive oxygen species production will be discussed with respect to cell death. This research will enhance our understanding of the contributions of both apoptosis and necrosis towards silica induced cell death and will aid in deducing a molecular pathway. Gaurav1.htmlshapeimage_18_link_0
Myosin motor function
The role of the motor protein myosin II in non-muscle cell physiology is still unclear. We have created light chain and heavy chain myosin mutants to examine their localization and function in chemotaxis and development. The results have shed new light on the distinction between myosin's role as an actin cross-linking protein and its role as a motor protein. We are currently collaborating with Dr. Juliet Lee's laboratory to measure the forces wild-type and mutant cells apply to surfaces during motility
Role of RKIP in motility
Raf Kinase Inhibitor Protein (RKIP) has been identified as a phosphatidylethanolamine-binding protein capable of inhibiting Raf-1 kinase, an enzyme significant in cell proliferation and cancer development.  When properly functioning, RKIP can mediate the expression of Raf-1 kinase and help prevent uncontrolled cell division.  RKIP also has suggested, but unclear, roles in spindle fiber formation during mitosis, regulation of apoptosis, and cell motility. The Fenteany laboratory in the Chemistry Department identified a new small molecule, named Locostatin, as a cell migration inhibitor in mammalian cells, with RKIP as its primary molecular target. Dictyostelium discoideum possess two RKIP proteins, RKIP-A and RKIP-B.  In order to begin to study the function of RKIP in D. discoideum and its role in cell motility, I created a mutant cell line which lacks a functional RKIP-A gene and showed that removal of RKIP-A does not affect vegetative motility, but impairs chemotaxis and development in the presence of drug.  Interestingly, RKIP-A knockout mutants appear more resistant to drug effects on vegetative motility than wild-type cells.  More research is needed to reconcile these seemingly contrasting results, and to better develop a model for RKIP-A’s role in cell motility.  Furthermore, a GFP-RKIP fusion protein and a double-knockout of RKIP-A and RKIP-B are being developed to help us better understand how this protein functions in processes involving cell migration.
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Silica-induced Phagocytosis 

Chronic inhalation of crystalline silica induces inflammation of the lungs, fibrosis, and subsequent development of silicosis. Although the inflammatory response of lung macrophages exposed to crystalline silica is well characterized, the molecular pathway of particle-cell interaction that lead to cellular toxicity are unclear. We have been studying the molecular pathway of non-opsonized (protein-coated or uncoated) silica and latex bead phagocytosis in order to understand how particles are internalized as well as the relationship of uptake to toxicity. 
Uptake of non-opsonized particles is slower than that of opsonized (antibody-coated) particles at 25°C, but occurs at nearly the same rate at 37°C. This data suggests that macrophages internalize non-opsonized particles by a temporally distinct pathway. The uptake of either crystalline or amorphous, spherical silica by macrophages induces cell death within 10 hours. Surprisingly, opsonizing silica to direct it to the Fc receptor-mediated phagocytosis pathway leads to a dramatic reduction in cell death. We hypothesize that this suppression of cell death is due to differences in particle phagosomal trafficking. 
Molecular markers involved during Fc receptor-mediated uptake are being used to examine this novel route of entry. Recent data suggests that, downstream of initial receptor recognition, the mechanism of non-opsonized particle uptake occurs similarly to this route. Moreover, inhibition of particle uptake by pre-treating cells with phagocytosis inhibitor drugs demonstrates that uptake is necessary to induce subsequent death. By understanding the mechanism of silica toxicity, it may be possible to devise ways to reduce its effects on human health. 
 
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The Roles of Actin Binding Proteins in cytoskeletal Organization and Function

Cellular motility plays a critical role in the processes of wound healing, morphogenesis, the immune response, and metastatic cancer.  The actin cytoskeleton and its associated proteins provide the mechanical force for membrane protrusion and motility of the cell.  The presence of actin-binding proteins is critical for the proper functioning of the cytoskeleton and all of the cell’s related activities.  Therefore, a thorough understanding of these proteins and how they interact with the cytoskeleton is essential.  While many actin-binding proteins have been identified, the mechanisms regulating their interaction with the cytoskeleton remain unclear.  We have previously shown that a particular domain on these proteins, termed the actin binding domain (ABD), is primarily responsible for directing the full protein to different cytoskeletal locations.  I am interested in determining what regulates the binding of these ABD’s to actin.  The ABD’s are very similar, yet data has shown them to localize drastically different from one another in the cell.  Using confocal microscopy, I will be determining spatial/temporal relationships between the ABP’s fimbrin, filamin, alpha-actinin, coronin, dynacortin, and others with the ultimate intention of creating a model that explains how these proteins cooperate to regulate cytoskeletal assembly.  Further, I will determine what roles affinity, phosphorylation, and cooperative binding play in determining when and where these proteins interact with the cytoskeleton. shapeimage_22_link_0
Differential Binding of α-Actinin and it’s domains to actin in polarized B16F1 cells


Calponin-homology family proteins bind actin through a conserved actin-binding domain(ABD), and would be expected to bind all forms of F-actin. In the Knecht lab, we examined the differential binding of α-actinin (an actin binding protein having an ABD domain in the N terminus, four spectrin like repeats and a pair of calcium binding EF hand domain in the C-terminus) and its domains relative to actin in lamellipodium of polarized B16F1 (mouse melanoma) cells. The full length protein which is known to form anti-parallel F-actin contractile bundles localizes to the proximal end of the lamellipodium and the filopodia. The actin binding domain (ABD) seems to be important for the localization of the protein to the structures like microspikes and filopodia in the lamellipodium and lamellar region. However the dimerisation of the ABD domain seems to restrict the localization of the protein to the proximal region of the lamellipodium and the microspikes similar to that of the full length protein. The differences in the localization of the monomeric and dimeric ABD could be due to the different binding affinities of the domains to F-actin. Total Internal Reflection Fluorescence (TIRF) microscopy is being used to study the difference in the binding of the different domains to F-actin. Using this technique, the rates of association (kon) and dissociation (koff) can be measured. The structural organization of the filaments and thereby the resultant architecture of the filaments due to the binding of the actin binding proteins like α-actinin can also be studied in vitro. 
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