Research Interests:

At its most basic, our work studies how the chemical reactions of biology are organized within the cellular boundaries. Biological organization is both spatial (cellular architecture), and temporal (cellular dynamics). We are interested in how cellular organization is maintained and harnessed by the animal to generate distinct physiological outputs, and how changes in intracellular organization underlie pathophysiological conditions.

Below you will find more details about ongoing projects in the lab.

Projects:

Action of Rab GTPases

We seek to understand the molecular detail behind the physiological process of exocytosis making use of model organisms as our experimental system. Exocytosis is the process by which membrane containers fuse with the plasma membrane of the eukaryotic cells. The membrane containers, or vesicles are lipid-enclosed compartments with selected membrane proteins embedded in the lipid bilayer and other soluble proteins and metabolites in the enclosed space. Exocytic events underlie diverse activities such the fast chemical transmission at the synapse, the release of insulin in response to signals and the expansion of the plasma membrane in preparation for cell growth and division.

The research goals of the lab center on how Ras-related GTPases regulate basic cellular processes. Ras superfamily members are found in all eukaryotic cells and function by cycling between GDP- and GTP-bound forms. It is the conformational, or shape change associated with the nucleotide binding state that enables these proteins to function as molecular switches. The crucial importance of this mechanism is demonstrated by the many cellular functions where GTPases are known to play pivotal roles and in diseases such as the retinal degeneration disease choroideremia where aberrant GTPase function abrogates normal cell growth and development.

In our studies, we are interested in understanding the mechanism of Rab protein activation: What are the cellular inputs and downstream consequences of Rab protein activation? How does Rab protein activation coordinate with other cellular events and what is the result of inappropriate activation? Towards this goal, we are using a variety of biochemical, molecular biological, cell biological and genetic techniques.

The diagram below summarizes the current model for the cycle of Rab function. Cytosolic GDP-bound protein is complexed to GDP dissociation inhibitor (GDI). After delivery to the target membrane, a guanine nucleotide exchange factor (GEF) catalyzes GDP/GTP exchange. After GTP hydrolysis, GDP-bound Rab is retrieved from the membrane by GDI. This cycle couples the events of membrane transport with the two nucleotide-bound states.

Role of Yip family members in membrane traffic

The YIP1 family is a group of small integral membrane proteins conserved throughout eukaryotes. Key features of this family are a domain organization consisting of a hydrophobic NH2-terminus, a hydrophilic COOH-terminus, the ability to bind di-geranylgeranylated Rab proteins and

Yip1p is a membrane protein which binds Rabs and is able to compete with GDI for Rab binding. Yip1p can dissociate the Rab-GDI heterodimer, enabling Rabs to reassociate with membranes and become activated. YIP1 is an essential gene that is highly conserved in evolution and is ubiquitously expressed in higher eukaryotes. Our goal is to exploit molecular genetics in yeast to gain information that will enable us to test the physiological roles of these factors in animal cells and we are developing methods to characterize the GDF activity of YIP1 in vitro and in vivo. We have identified Yip1p-related proteins in mice and humans which we will use to explore the hypothesis that Rab membrane recruitment in both yeast and mammalian cells is mediated by homologous strategies. Interestingly, we have discovered that Yip1p is capable of interaction with many different Rab proteins. These data place the role of Yip1p as the mirror image of the role played by GDI which is also a pleiotropic factor. However, while GDI removes Rabs from membranes into the cytosol, Yip1p acts to retrieve Rabs from the cytosol back onto membranes. The importance of GDI is well known and is crucial for normal cellular development, for example, mutations in GDI are responsible for non-specific X-linked mental retardation. Studies of Yip1p will provide a deeper understanding of the mechanism of GDI action and Rab membrane recruitment.

Role of Elp1p in the neurological disease Familial dysautonomia (FD)

One of the remarkable features of eukaryotic cells is the elaborate system of membrane-enclosed intracellular organelles and the continuous flow of material between organelles in the secretory and endocytic pathways. The major focus of membrane traffic over the past 20 years has related to the machinery that constructs and fuses vesicles. In our lab, we have conducted screens to discover how the basic machinery of membrane transport is regulated; to understand how cells respond to the signaling cascades generated by physiological stimuli at the level of membrane traffic to regulate the organelle identity and cellular architecture that ultimately impacts tissue and organ function. We identified the gene ELP1 as a negative regulator of polarized secretion. This discovery has immediate implications for the human autosomal recessive disease Familial Dysautonomia, (FD; Riley-Day syndrome. FD is a congenital sensory neuropathies characterized by progressive depletion of sensory and autonomic neurons, a degeneration which is thought to start early in fetal life. Mutations in human ELP1 (IKAP) are responsible for Familial Dysautonomia (FD), however the underlying molecular mechanisms by which these mutations cause FD are unknown. Our research findings suggested a rationale for the pathophysiology of FD due to a dysfunction of exocytosis in neurons.

Bioinformatic studies of Rab GTPases

Researchers looking to solve biological problems have access to enormous amounts of sequence information and the desktop computational infrastructure to personally interrogate and analyze large datasets. Many powerful bioinformatics tools are available online, however this discourages the customized analysis of data that is necessary for the experimental scientist to make maximally effective use of the information. In addition, a customized environment facilitates the critical evaluation of bioinformatic methods. We have developed a protocol developed to aid in classification of subfamilies and subclasses of a superfamily using the personal desktop computer. The visual representation of the qualitative and quantitative results of data analyses is also considered. To date we have focused on Rab GTPases but our algorithms are more widely applicable to the classification of any given protein family.

Subcellular localization of yeast Rab proteins

The yeast Saccharomyces cervisiae is a powerful organism for genetic and biochemical studies. For cell biology applications, the subcellular localization of yeast cells by light microscopy has historically been compromized by its size and shape. The small size of the yeast cell limits spatial resolution in the x-y plane and the depth of the cell in the z-dimension results in blurring due to out-of-focus signal in fluorescence applications. We are using deconvolution microscopy to improve spatial resolution. Using a motorized microscope stage, serial images are captured as a z-stack through the cell (typically 0.1-0.2 um per slice). The use of a highly sensitive CCD camera with electron multiplication gain minimizes photobleaching and allows aquistion of weak signals. This set of images is then subjected to double-blind deconvolution, using a dual-Xeon workstation to minimize processing time. Images can then be rotated through 360 degrees and be subjected to further processing (e.g. isosurface rendering) or viewed as a QuickTime movie to gain maximum information on the localization of the protein in three dimensions.

---