Kenneth E. Prehoda - US grants

Our overall goal is to understand the molecular basis of cell movement. Improperly regulated cell movement is involved in many diseases states including metastatic cancer and inflammation. Cell movement involves the remodeling of the actin cytoskeleton in response to external cues. We are studying a protein that transduces signaling information to the actin cytoskeleton. This protein, N-WASP, appears to act as a switch that, given the appropriate upstream signals, targets and activates the actin polyrnerization machinery at the proper sites. To understand how N-WASP is localized , we are examining it's N-terminal domain, which is thought to be critical for targeting. We are attempting to identify ligands for the domain and to characterize ligand specificity. We will follow it's localization in vivo in chemotaxic cells using domains fused to green fluorescent protein. To understand how N-WASP acts as a molecular switch, we are attempting to identify and characterize repressive intramolecular interactions and to determine the structure of N-WASP alone and in the presence of activating ligands such as CDC42.

The long-term goal of this work is to understand the structural and biochemical basis of cell polarity. Polarity is a fundamental property of cells that is required for proper development as well as adult physiology. For example, during development, cell fate determinants are polarized in dividing cells as a mechanism for generating cell type diversity. For spatially and temporally precise establishment of cell polarity to occur, cellular signals must be interpreted and ultimately coupled to the segregation of relevant cellular components. We have chosen to focus on a protein complex that plays a central role in controlling cell polarity. This complex, which includes the PAR proteins Par-3 and Par-6, and the atypical protein kinase C (aPKC), localizes to specific cellular sites and subsequently is thought to recruit downstream components of the cell polarization machinery. We have chosen to focus on the proteins in this evolutionarily conserved complex in order to understand their interactions, how they are localized, and how they act as switches to transmit the information that controls cell polarity. Much of our effort will be directed towards understanding how the Rho GTPase family of signaling molecules, which have recently been shown to interact with Par-6, regulates localization, assembly, and/or activity of these adapter proteins. We are also focusing on elucidating how the PDZ protein-interaction domain mediates assembly of the complex and interactions with downstream components. The remainder of our effort will be directed at understanding how these proteins subsequently activate components of the cell polarity machinery. We will use a combined biochemical, biophysical and cell biological approach to investigate Par complex function. Using biochemical and biophysical methods, we will examine the physical basis by which the Par complex assembles and interacts with signaling molecules and downstream components of the cell polarity machinery. Finally, we will test the in vivo importance of these interactions in Drosophila epithelial and neuroblast cells, two models for cell polarity.

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the molecular basis of regulated mitotic spindle orientation. Alignment of the mitotic spindle along a predetermined axis is required for proper cell function in many contexts, including differentiation, embryogenesis, and organogenesis. For example, during the asymmetric division of Drosophila neuroblasts, precursors of the central nervous system, cell fate determinants localize to opposite poles of the cell such that they become segregated into discrete daughter cells. Proper distribution of determinants, and subsequent fate specification, requires that the mitotic spindle align precisely with the polarity axis. We propose to investigate this process by reconstituting spindle orientation in a cultured cell that does not normally orient the spindle. Establishing regulated spindle positioning in this context will allow us to determine which components are sufficient for spindle orientation, and we propose to examine these components biochemically and structurally to determine their mechanism of action. In our preliminary work we have successfully polarized Drosophila S2 cells using the adhesion protein Echinoid and have found that expression of Echinoid proteins in which the cytoplasmic portion has been replaced with domains from the Partner of Inscuteable (Pins) protein can robustly orient the spindle in a manner similar to neuroblasts. We are using a combination of biochemical, biophysical, and cell biological methods to investigate the function of molecules that we identify in our spindle orientation reconstitution, including Pins. PUBLIC HEALTH RELEVANCE: During cell division, chromosomes are separated into the daughter cells by the mitotic spindle. In many cells, the spindle must be precisely positioned for proper tissue organization, differentiation, or prevention of tumor formation. In this work, we are attempting to identify the cellular machinery required for spindle position control by attempting to recreate this process in a cell type that does not normally orient its spindle. As the loss of accurate spindle positioning is associated with human disease, improving our understanding of the molecules that control this process will contribute to our knowledge of the mechanisms of disease states.

Project abstract Funding is requested for the purchase of an AKTA pure 25 protein puri?cation system for purifying the reagents necessary for examining the biochemical activity of the Par complex, a key aspect of the parent grant, R35GM127092. To reconstitute and interrogate Par complex proteins to understand the mechanism by which polarity is regulated, highly pure reagents are required. This is challenging for two reasons. First, the Par complex itself is di?cult to obtain as it is obtained from transiently transfected cultured cells. Second, our work requires making various modi?ed forms of Par complex proteins (e.g. truncations) and this instrument will allow us to separate them from impurities with high resolution. We expect that the additional capabilities provided by this instrument will have a signi?cant and sustained positive impact on our research program by increasing the reliability and reproducibility of key reagents.

Project abstract! Funding is requested for the purchase of a Bruker Micro?ex SMART LS MALDI-TOF mass spectrometer for verifying the reagents we produce. The instrument will be used by my research group and by my colleague Brad Nolen who has submitted a cross-referenced application. In R35GM127092, the parent grant for this supplement application, my laboratory proposes to uncover the mechanism of animal cell polarization using the neuroblast as a model system. A key aim is to use reconstituted puri?ed Par complex proteins to understand the mechanism by which polarity is regulated. While we routinely use gel electrophoresis as a method for determining the identity and purity of our reagents, several features of our system make mass spectrometry an important complement to this standard technique. First, a component of the Par complex, atypical Protein Kinase C, migrates abnormally by gel electrophoresis because of essential post translational modi?cations. Second, our work requires making various modi?ed forms of Par complex proteins (e.g. truncations) and this instrument will allow us to verify them with high resolution mass information. We expect that the additional information provided by this instrument will have a signi?cant and sustained positive impact on our research program by increasing the reliability and reproducibility of key reagents.