Adam Vincent Kwiatkowski

• Ph.D.
• Professor (Associate) at University of Pittsburgh

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular p...

Spatial and temporal protein tracking in live cells permits proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially ma...

α-Catenin binds directly to β-catenin and connects the cadherin–catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interact...

α-catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of αT(t...

The adherens junction (AJ) couples the actin cytoskeletons of neighboring cells to allow mechanical integration and tissue organization. The physiological demands of intercellular adhesion require that the AJ be responsive to dynamic changes in force while maintaining mechanical load. These demands are tested in the heart, where cardiomyocyte AJs m...

αT (Testes)-catenin, a critical factor regulating cell–cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disc (ICD), a unique cell-cell junction that couples cardiomyocytes. Loss of αT-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since αT-catenin i...

The adherens junction (AJ) couples the actin cytoskeletons of neighboring cells to allow mechanical integration and tissue organization. The physiological demands of intercellular adhesion require that the AJ be responsive to dynamic changes in force while maintaining mechanical load. These demands are tested in the heart, where cardiomyocyte AJs m...

The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signa...

Top 500 DE-cad ERC hits. (XLSX)

Enrichment analysis of DE-cad ERC human orthologs: Canonical pathways. (XLSX)

RNAi stocks used in this study. (XLSX)

Border cell migration in raskol RNAi egg chambers. (AVI)

DE-cadherin levels at BC cell-cell contacts A-F. Representative images of BC clusters expressing DE-cad-GFP and UAS-RNAi constructs (A-E) or UAS-RFP (F) in BCs under the control of slbo-GAL4. DAPI (first column; blue in merge), DE-cad-GFP (second column; green in merge), F-actin (third column; red in merge) and merge (fourth column) channels shown....

Raskol colocalizes with DE-cad in the amnioserosa and ectodermal cells during DC. Time-lapse images of embryos expressing Raskol-YFP and DE-cad-RFP during DC. Raskol colocalizes with DE-cad at cell-cell contacts in the amnioserosa (arrows). Raskol colocalizes with DE-cad at the zippering interface of the dorsal-most ectodermal cells (arrowheads). I...

Enrichment analysis of DE-cad ERC human orthologs: Disease. (XLSX)

Border cell migration in control RNAi egg chambers. Lifeact-GFP and RNAi transgenes expressed under control of slbo-GAL4. 30 mins. (AVI)

Border cell delamination defects in raskol RNAi egg chambers. (AVI)

Border cell cluster disassociation defects in raskol RNAi egg chambers. (AVI)

Raskol localization and expression analysis. A. Mean cytoplasmic levels of Raskol in PCs and BCs relative to NCs. Cytoplasmic levels of Raskol were significantly higher in PCs compared to BCs according to Welch’s t-test (n = 58, p<0.0001). B. shg and raskol expression patterns display similar trends during embryonic development. RNA-seq based expre...

Border cell migration and cluster disassociation data. (XLSX)

Raskol colocalizes with DE-cad in the FE. A-B. Egg chambers expressing Raskol-YFP (green) and stained for DE-cad (red) and F-actin (blue). Raskol is enriched at the FE apical surface where it colocalizes with DE-cad and F-actin (arrows). Individual channels correspond to the outlined box in the merged image. A. Dorsal view of an egg chamber. B. Cro...

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte A...

The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link cortical actin to intercellular adhesions. Over time, evolutionary pressures have shaped...

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte A...

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to...

Cardiomyocytes are joined end-to-end by a complex adhesive structure known as the intercalated disc (ICD). The ICD is composed of mechanical and electrical junctional complexes including the adherens junction (AJ). The core of the AJ is the cadherin/catenin complex, which links the actin cytoskeletons of neighboring cells to provide mechanical cont...

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique – the actin co-sedimentation, or pelleting, assay – to determine whether a protein or protein domain binds F-actin and to measure...

Intercellular epithelial junctions formed by classical cadherins, β-catenin and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and...

α-Catenin is the primary link between the cadherin/catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin/cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin/catenin complex, αE-catenin requires force to bind F-acti...

Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cue...

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both...

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell-cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments, and that binding is accompanied by a c...

It is unknown whether homologs of the cadherin·catenin complex have conserved structures and functions across the Metazoa. Mammalian αE-catenin is an allosterically regulated actin-binding protein that binds the cadherin·β-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional propert...

The regulated assembly and organization of actin filaments allows the cell to construct a large diversity of actin-based structures specifically suited to a range of cellular processes. A vast array of actin regulatory proteins must work in concert to form specific actin networks within cells, and spatial and temporal requirements for actin assembl...

The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved....

alphaE-catenin binds the cell-cell adhesion complex of E-cadherin and beta-catenin (beta-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of alphaE-catenin to the E-cadherin-beta-cat complex lowers alphaE-catenin affinity for F-actin, and alphaE-catenin alone can bind F-actin and inhibit Arp2/3 complex-mediated actin polym...

CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal micros...

A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based met...

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VA...

Mammalian cortical development involves neuronal migration and neuritogenesis; this latter process forms the structural precursors to axons and dendrites. Elucidating the pathways that regulate the cytoskeleton to drive these processes is fundamental to our understanding of cortical development. Here we show that loss of all three murine Ena/VASP p...

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell-cell and cell-matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we esta...

Synapses of the central nervous system (CNS) are specialized cell-cell junctions that mediate intercellular signal transmission from one neuron to another. The directional nature of signal relay requires synaptic contacts to be morphologically asymmetric with distinct protein components, while changes in synaptic communication during neural network...

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses(2-10). But the resistance of important cell types...

Tuba is a 178kD protein containing four NH2-terminal SH3 domains, a central Dbl homology (DH) domain followed by a BAR domain, and two COOH-terminal SH3 domains. The four NH2-terminal SH3 domains bind the GTPase dynamin, a protein critical for the fission of endocytic vesicles. The DH domain functions as a CDC42-specific guanine nucleotide exchange...

Tuba is a novel scaffold protein that functions to bring together dynamin with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Tuba binds a variety of actin regulatory proteins, including N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1, and Ena/VASP prote...

Regulation of cytoskeletal dynamics is required to coordinate cell movement, adhesion and shape change. The Ena/VASP protein family is thought to play an important role in linking signaling pathways to remodeling of the actin cytoskeleton. This review will examine the mechanisms by which Ena/VASP function might control actin dynamics and how these...

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to tra...

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to tra...

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, an...

Proteins of the Ena/VASP family are implicated in processes that require dynamic actin remodeling such as axon guidance and platelet activation. In this work, we explored some of the pathways that likely regulate actin dynamics in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, were highly expressed in hematopoietic cells of thym...

Rhodobacter sphaeroides 2.4.1, which is incapable of denitrification, has been found to carry nnrR, the nor operon, and nnrS, which are utilized for denitrification in R. sphaeroides 2.4.3. The gene encoding nitrite reductase was not found in 2.4.1. Expression of beta-galactosidase activity from a norB-lacZ fusion was activated when cells of 2.4.1...

Nitrite reductase catalyzes the reduction of nitrite to nitric oxide, the first step in denitrification to produce a gaseous product. We have cloned the gene nirK, which encodes the copper-type nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides, strain 2.4.3. The deduced open reading frame has significant identity with other c...

During denitrification, freely diffusible nitric oxide (NO) is generated for use as a terminal electron acceptor. NO is produced by nitrite reductase (Nir) and reduced to nitrous oxide by nitric oxide reductase (Nor). Using Nir and Nor-deficient mutants of Rhodobacter sphaeroides 2.4.3, we have shown that the endogenous production of NO or the addi...