The basic cell biology of transport of lipids and proteins in cells will be studied using quantitative fluorescence microscopy coupled with biochemical and ultrastructural tools. The proposed studies build directly on previous work establishing the basic methods to be used. The proposed work Is divided in two main themes relating to membrane protein transport (Aims l-lll) and cholesterol and lipid transport (Aims IV and V). These two themes interact synergistically because proteins are responsible for lipid transport, and membrane lipids strongly Influence the transport and distribution of membrane proteins.
In Aim I the role of specific proteins will be studied using rapid and selective protein inactivation by chemical crosslinking or other methods. The effect bf inactivating target proteins (e.g, clathrin, GGAs, or Rab proteins) will be studied by following the transport of specific marker proteins (e.g., labeled Tac-Furin, Tac-TGN38, CI-MPR) within minutes after inactivation.
In Aim II the role of Clc-7 and Ostml In regulating lysosomal pH will be studied. Delivery of these proteins to late endosomes and lysosomes is essential for proper acidification of these organelles In microglial cells. The role of these proteins in organelle acidification in other cell types, Including dendritic cells, will be examined. The transcriptional regulation of Clc-7 and Ostml In microglia and other cell types will also be examined.
In Aim III the ability of macrophages and other cell types to create functional extracellular lysosomes will be examined. These lysosomal synapses play an important role In degradation of extracellular digestion of lipoprotein deposits, and their role in other types of extracellular degradation will be examined.
In Aim I V intracellular sterol transport and distribution will be characterized using fluorescent sterols and biochemical analyses. Although proteins such as ABCA1 and ABCG4 are essential for export of cholesterol to HDLs, the precise role of these proteins remains unclear. The effect of expression of these proteins on the transbilayer distribution of sterol at the plasma membrane will be determined using previously developed fluorescence quenching assays. Similarly, the effect of NPC1 and NPC2 on the transbilayer distribution of sterol in late endosomes will be examined. The mechanisms and function of high levels of nonvesicular sterol transport within cells will also be'examined.
In Aim V the Interplay between sterols and membrane traffic will be explored. In particular, the function of tubules that emanate from late endosomes In response to transient sterol loading will be studied.

Public Health Relevance

We are studying the basic mechanisms by which proteins and lipids move within cells. Findings from these studies are directly related to diseases associated with disorders in lipid and cholesterol transport, including diabetes, atherosclerosis, and lysosomal storage disorders. Our studies of protein degradation may also be related to diseases associated with abnormal protein deposits, including Alzheimer's disease and other amyloid-associated diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK027083-35
Application #
8448330
Study Section
Special Emphasis Panel (NSS)
Program Officer
Haft, Carol R
Project Start
1980-04-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
35
Fiscal Year
2013
Total Cost
$619,424
Indirect Cost
$249,545
Name
Weill Medical College of Cornell University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Maxfield, Frederick R (2014) Role of endosomes and lysosomes in human disease. Cold Spring Harb Perspect Biol 6:a016931
Haka, Abigail S; Grosheva, Inna; Singh, Rajesh K et al. (2013) Plasmin promotes foam cell formation by increasing macrophage catabolism of aggregated low-density lipoprotein. Arterioscler Thromb Vasc Biol 33:1768-78
Helquist, Paul; Maxfield, Frederick R; Wiech, Norbert L et al. (2013) Treatment of Niemann--pick type C disease by histone deacetylase inhibitors. Neurotherapeutics 10:688-97
Maxfield, Frederick R; Wustner, Daniel (2012) Analysis of cholesterol trafficking with fluorescent probes. Methods Cell Biol 108:367-93
Majumdar, Amitabha; Capetillo-Zarate, Estibaliz; Cruz, Dana et al. (2011) Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes. Mol Biol Cell 22:1664-76
Pagler, Tamara A; Wang, Mi; Mondal, Mousumi et al. (2011) Deletion of ABCA1 and ABCG1 impairs macrophage migration because of increased Rac1 signaling. Circ Res 108:194-200
Mesmin, Bruno; Pipalia, Nina H; Lund, Frederik W et al. (2011) STARD4 abundance regulates sterol transport and sensing. Mol Biol Cell 22:4004-15
Rosenbaum, Anton I; Maxfield, Frederick R (2011) Niemann-Pick type C disease: molecular mechanisms and potential therapeutic approaches. J Neurochem 116:789-95
Pipalia, Nina H; Cosner, Casey C; Huang, Amy et al. (2011) Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts. Proc Natl Acad Sci U S A 108:5620-5
Maxfield, Frederick R; van Meer, Gerrit (2010) Cholesterol, the central lipid of mammalian cells. Curr Opin Cell Biol 22:422-9

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