Regulation of contractile vacuole formation and activity in Dictyostelium

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV–plasma membrane fusion and discharging. disgorgin− cells produce large CVs due to impaired CV–plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin− large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function

Multivariate profiling of neurodegeneration-associated changes in a subcellular compartment of neurons via image processing

<p>Abstract</p> <p>Background</p> <p>Dysfunction in the endolysosome, a late endosomal to lysosomal degradative intracellular compartment, is an early hallmark of some neurodegenerative diseases, in particular Alzheimer's disease. However, the subtle morphological changes in compartments of affected neurons are difficult to quantify quickly and reliably, making this phenotype inaccessible as either an early diagnostic marker, or as a read-out for drug screening.</p> <p>Methods</p> <p>We present a method for automatic detection of fluorescently labeled endolysosomes in degenerative neurons in situ. The Drosophila <it>blue cheese </it>(<it>bchs</it>) mutant was taken as a genetic neurodegenerative model for direct in situ visualization and quantification of endolysosomal compartments in affected neurons. Endolysosomal compartments were first detected automatically from 2-D image sections using a combination of point-wise multi-scale correlation and normalized correlation operations. This detection algorithm performed well at recognizing fluorescent endolysosomes, unlike conventional convolution methods, which are confounded by variable intensity levels and background noise. Morphological feature differences between endolysosomes from wild type vs. degenerative neurons were then quantified by multivariate profiling and support vector machine (SVM) classification based on compartment density, size and contrast distribution. Finally, we ranked these distributions according to their profiling accuracy, based on the backward elimination method.</p> <p>Results</p> <p>This analysis revealed a statistically significant difference between the neurodegenerative phenotype and the wild type up to a 99.9% confidence interval. Differences between the wild type and phenotypes resulting from overexpression of the Bchs protein are detectable by contrast variations, whereas both size and contrast variations distinguish the wild type from either of the loss of function alleles <it>bchs1 </it>or <it>bchs58</it>. In contrast, the density measurement differentiates all three <it>bchs </it>phenotypes (loss of function as well as overexpression) from the wild type.</p> <p>Conclusion</p> <p>Our model demonstrates that neurodegeneration-associated endolysosomal defects can be detected, analyzed, and classified rapidly and accurately as a diagnostic imaging-based screening tool.</p