Research Description

The central vacuolar system of eukaryotic cells is compartmentalized into distinct membrane-bound organelles and tubulovesicular structures, each with its own characteristic function and set of protein constituents. Work in my laboratory is focused on understanding how integral membrane protein complexes are assembled and sorted to the appropriate compartments within the late secretory and endocytic pathways, and how sorting and assembly contribute to the biogenesis of several cell type-specific organelles and to the regulation of immunity and hemostasis.

Our primary focus over the past 20 years has been on melanosomes of pigmented cells. Melanosomes are unique lysosome-related organelles present only in cells that make melanin, the major synthesized pigment in mammals. Melanosomes are among a number of tissue-specific lysosome-related organelles (LROs) that are malformed and dysfunctional in a group of rare heritable disorders, including Hermansky-Pudlak and Chediak-Higashi syndromes. Moreover, mutations in genes encoding pigment cell-specific proteins underlie various forms of non-syndromic oculocutaneous and ocular albinism. In an effort to understand the molecular basis of these diseases, we are dissecting the molecular mechanisms that regulate how different stage melanosomes are formed and integrated with the endosomal pathway, and how specific non-enzymatic melanosome components contribute to melanin synthesis.  We use biochemical, morphological, and genetic approaches to follow the fates of melanosome-specific and ubiquitous endosomal and lysosomal proteins within pigment cells from normal individuals or mice and disease models. Using these approaches, we are (1) detailing protein transport pathways that lead to the formation of these unusual organelles, (2) dissecting biochemical pathways that lead to their morphogenesis, (3) defining how these processes are subverted by genetic disease, and (4) understanding how genetic variation impacts skin pigmentation. Current efforts focus on how factors that are deficient in patients and mouse models of Hermansky-Pudlak syndrome impact melanosome biogenesis. These factors interact with classical components of the membrane trafficking machinery such as SNAREs and coats, and we are dissecting how these interactions result in the delivery of cargoes to unique organelle structures in these cells. We are particularly interested in the formation of tubular connections between endosomes and maturing melanosomes, as several factors that are disrupted in Hermansky-Pudlak syndrome impact the formation and/or dynamics of these transport carriers. We are also focused on how a number of genes impact pigmentation by altering the lumenal pH of melanosomes or of lysosomes that interact transiently with melanosomes inside melanocytes.

Because genetic diseases like Hermansky-Pudlak syndrome affect multiple organ systems, we are dissecting how similar sorting processes involved in melanosome biogenesis influence other organelles in different cell types. The first involves LROs in platelets called dense granules and alpha granules. Dense granules store adenine nucleotides, polyphosphate, serotonin and calcium that are released upon platelet activation and are required for optimal blood clotting. Like melanosomes, dense granules are malformed in Hermansky-Pudlak syndrome. In collaboration with a variety of investigators at CHOP and Penn Medicine, we are studying how dense granule contents are delivered within platelets and their precursors (megakaryocytes) and how these processes are altered in Hermansky-Pudlak syndrome and other bleeding disorders. Additional collaborative studies focus on the formation and protein targeting to alpha granules, which are distinct LROs in platelets that store secretory protein contents. In collaboration with Susan Guttentag at Vanderbilt, we are extending our knowledge of melanosome biogenesis to the lamellar bodies of alveolar type 2 cells in the lung. These LROs synthesize and store surfactant, and are also disrupted in several forms of Hermansky-Pudlak syndrome. We are identifying lamellar body components and the pathways by which they access nascent lamellar bodies. A third cellular system is the dendritic cell, a master regulator of T cell immune function. We have found that in dendritic cells from a mouse model of Hermansky-Pudlak syndrome type 2, phagocytosed particles elicit impaired signaling to activate innate immune functions, with consequent impairment in activating CD4+ T cells of the adaptive immune system against particulate invaders such as bacteria. We are trying to understand the molecular bases of these effects and their link to antigen presentation.

Finally, melanosome precursors harbor intralumenal fibrils upon which melanins deposit in later stages. The main component of these fibrils is a pigment cell-specific protein, PMEL. Fibrils formed by PMEL in vitro resemble amyloid formed in neurodegenerative diseases such as Alzheimer, Parkinson, and prion diseases. We hope that by dissecting how PMEL forms amyloid under physiological conditions, we may not only understand melanosome biogenesis but also the formation of amyloid under pathological conditions.

Diversity & Inclusion Initiatives

• Trainer, PennPORT program (two post-doctoral fellows, 2010-2018)
• Trainer, Summer Undergraduate Internship Program (1997-2020)
• Trainer, HHMI summer undergraduate program (1998)
• Trainer, Univ. of Pennsylvania diversity undergraduates (1996-2020)
• ACT Discussion leader