Browsing by Subject "Overexpression"
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Item Functional analysis of Abp1 in Dictyostelium(2006-08) Wang, Yanqin, 1974-; O'Halloran, TheresaThis work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis.Item Understanding the mechanisms of floor plate specification in the vertebrate midbrain and its functions during development(2009-08) Bayly, Roy Downer, 1981-; Agarwala, SeemaWe have previously shown that the arcuate organization of cell fates within the ventral midbrain critically depends upon the morphogen, Sonic Hedgehog (SHH), which is secreted from a signaling center located along the ventral midline, called the floor plate (FP). Thus, it is ultimately the specification of the FP that is responsible for the patterning and specification of ventral midbrain cell fates. Interestingly, we have found that the chick midbrain FP can be divided into medial (MFP) and lateral (LFP) regions on the basis of gene expression, mode of induction and function. Overexpression of SHH alone is sufficient to recapitulate the entire pattern of ventral cell fates, although remarkably it cannot induce MFP, consistent with the observation that the MFP is refractory to any perturbations of HH signaling. In contrast, overexpression of the winged-helix transcription factor FOXA2/HNF3[beta]robustly induced the MFP fate throughout ventral midbrain while blocking its activity resulted in the absence of the MFP. Thus, by analyzing the differences between SHH and FOXA2 blockade and overexpression, we were able to attribute functions to each the LFP and the MFP. Notably, we observed that FOXA2 overexpression caused a bending of the midbrain neurepithelium that resembled the endogenous median hinge-point observed during neurulation. Additionally, FOXA2 misexpression led to a robust induction of DA progenitors and neurons that was never observed after SHH expression alone. In contrast, we found that all other ventral cell types required HH signaling directly, at a distance and early on in the development of the midbrain when its tissue size is relatively small. Additionally, HH blockade resulted in increased cell-scatter of the arcuate territories and in the disruption of the regional boundaries between the ventral midbrain and adjacent tissue. Thus, we bring new insight into the mechanism by which midbrain FP is specified and ascribe functional roles to its subregions. We propose that while the MFP regulates the production of dopaminergic progenitors and the changes in cellshape required for bending and shaping the neural tube, the LFP appears to be largely responsible for cell survival and the formation of a spatially coherent pattern of midbrain cell fates.