Biomedical subjects

Sushmita Mukherjee

Publications and source records attributed to Sushmita Mukherjee.

6 recordsLinked to original sources

PubMed2006

Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect.

We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.

Animals↗

PubMed2006

Endocytic recycling compartments altered in cisplatin-resistant cancer cells.

The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling compartment (ERC) and found that the distribution of the ERC is altered in KB-CP.5 cells compared with parental KB-3-1 cells. A tightly clustered ERC is located near the nucleus in parental KB-3-1 cells but it appears loosely arranged and widely dispersed throughout the cytoplasm in KB-CP.5 cells. The altered distribution of the ERC in KB-CP.5 cells is related to the amount and distribution of stable detyrosinated microtubules (Glu-alpha-tubulin), as previously shown in Chinese hamster ovary B104-5 cells that carry a temperature-sensitive Glu-alpha-tubulin allele. In addition, B104-5 cells with a dispersed ERC under nonpermissive conditions were more resistant to cisplatin compared with B104-5 cells with a clustered ERC under permissive conditions. We conclude that resistance to cisplatin might be due, in part, to reduced uptake of cisplatin resulting from an endocytic defect reflecting defective formation of the ERC, possibly related to a shift in the relative amounts and distributions of stable microtubules.

Animals↗

PubMed2004

Selecting transpositions using phage P1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and P1-derived artificial chromosomes.

New Tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs). Each contained a wild-type loxP site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of DNA. In contrast to loxP transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering with those endogenous to the BAC clone in functional mapping studies. Progressive deletions from the loxP end of genomic DNA were efficiently generated using these transposons, and a series of truncations generated in a green fluorescence protein (GFP)-BAC fusion clone unambiguously identified three new long-range enhancer sequences functionally in the Nkx2-5 gene in transgenic mice. Insertions of these new transposons lacking antibiotic resistance genes into a BAC or PAC were indirectly selected by their ability to delete enough DNA from the clone so as to enable its packaging within a P1 phage head with both loxP sites intact for subsequent recovery of the large plasmid. The outcome of such an indirect mode of selection is both desirable and undesirable. First, because the screen is not antibiotic resistance marker dependent, the same transposon can be used to generate nested deletions efficiently in both BACs and PACs. Second, deletions through intrainsert recombinations unrelated to loxP/Cre also get packaged and recovered, and size analyses of the BAC/PAC vector band after NotI digestion is indispensable to identify authentic loxP/Cre deletions. The procedure nevertheless offers a potential approach to map recombinogenic sequences in BACs and PACs.

Animals↗

PubMed2004

Lipid and cholesterol trafficking in NPC.

Niemann-Pick type C, or NPC for short, is an early childhood disease exhibiting progressive neurological degeneration, associated with hepatosplenomegaly in some cases. The disease, at the cellular level, is a result of improper trafficking of lipids such as cholesterol and glycosphingolipids (GSLs) to lysosome-like storage organelles (LSOs), which become engorged with these lipids. It is believed that the initial defect in trafficking, whether of cholesterol or a GSL, results in an eventual traffic jam in these LSOs. This leads to the retention of not only other lipids, but also of transmembrane proteins that transiently associate with the late endosomes (LE) in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN). In this review, we discuss the biophysical properties of lipids and cholesterol that might determine their intracellular itineraries, and how these itineraries are altered in NPC cells, which have defects in the proteins NPC1 or NPC2. We also discuss some potential therapeutic directions being suggested by recent research.

Animals↗

PubMed2004

Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes.

Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC(12) and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC(16) and DiIC(18)) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC(16), were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes.

Animals↗

PubMed2004

Membrane domains.

Considerable evidence shows that lateral inhomogeneities in lipid composition and physical properties exist in biological membranes. These membrane lipid domains are proposed to play important roles in processes such as signal transduction and membrane traffic. However, there is not at present an adequate description of the nature of these lipid domains in terms of their size, abundance, composition, or dynamics. We discuss the current analyses of the properties and function of membrane domains in cells and compare their properties with chemically simpler model membrane systems that can be understood in greater detail.

Lipid Metabolism↗