People in the Rothman Lab

We have been using functional genetic screens of cell- based assays to understand how cells eliminate accumulated and aggregated proteins. A common feature in a wide array of progressive neurodegenerative diseases, protein accumulation and aggregation are often a by-product of distinct genetic mutations. Interestingly, in several conditional mouse models of neurodegenerative diseases such as the triplet repeat disorder Huntington's disease (HD), as well as Parkinson's disease and prion disease, clearance of these proteins lead not only to a halt of symptomatic progression but also to a regression of the disease.

Rather than starting with a hypothesis driven approach, we have used a two-tier genetic screen using Affymetric gene arrays followed by siRNAs to determine possible regulators of mutant protein accumulation and clearance. The patterns of hits generated from the screen implicates the lysosome-mediated degradation pathway, macroautophagy. We are currently pursuing the hits to elucidate regulators and modifiers of this pathway.

Flipped SNAREs fuse whole cells, and provide a system where the role of each membrane fusion component can quantitatively be individually determined. Additionally, these cells are large (~200 µM), and can provide robust electrophysiological capacitance step and conductance data upon membrane fusion (Lindau and Gomperts 1991; Lollike and Lindau 1999). These features make flipped SNAREs an ideal system to deconstruct and define the roles of the SNARE fusion machinery and companion proteins, while providing quantitative kinetic measurements of their effects.

In regulated exocytosis, the core membrane fusion machinery-SNARE proteins-cooperate with a group of regulatory factors to couple membrane fusion to particular stimuli, such as an increase of intracellular calcium ion concentration. I utilize in vitro reconstitution to ask mechanistic questions concerning precisely how regulatory proteins control exocytosis at the molecular level. Our long-term vision is to reconstitute, protein by protein, the basic properties and fine-tuning of regulated exocytosis.

The energy made available by the directed assembly of the SNARE complex drives membrane fusion. I am determining these nanoscale forces at the single molecule level with a variety of approaches including the Surface Force Apparatus, the Vesicle Micromanipulation technique and the Cell Micromanipulation technique^.

Melanocortins are a group of peptide hormones expressed primarily in the pituitary and arcuate nucleus of the hypothalamus and have numerous roles in the central and peripheral nervous systems. Five melanocortin receptors have been identified to date, all of which are type A G-protein coupled receptors (GPCRs). Of these receptors, the melanocortin receptor subtype 4 (MC4-R) plays an important role in the mechanism regulating appetite and body weight. Mutations in the MC4-R cause obesity in both mice and humans.  Energy homeostasis through this pathway is highly susceptible to quantitative variation in MC4-R expression. We are currently using a cell-based approach to study how changes in steady-state regulation of MC4-R function could be involved in the disregulation of the melanocortin signaling pathway, and the role of vesicle trafficking in these processes. To do that, we use the mouse hypothalamic GT1 cell line, a neurosecretory cell line produced as a neuronal tumor in transgenic mice. In this cell line, MC4-R is endogenously expressed and its activation couples to gonadotropin-releasing hormone release. By gene expression profiling, we expect to have an insight into the mechanisms that regulate MC4-R function. To complement this approach, we are also currently developing stable cell lines expressing molecular-tagged receptor to perform genetic and chemical screenings.