Showing papers by "Dick Hoekstra published in 2003"

Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains.

Abstract: In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.

Membrane Dynamics and the Regulation of Epithelial Cell Polarity

Abstract: Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains and distinct intracellular compartments relevant to cell polarity development, have triggered extensive research on issues that focus on how the polarity is generated and maintained. Apart from proper assembly of tight junctions, their potential functioning as landmark for the transport machinery, cell-cell adhesion is obviously instrumental in barrier formation. In recent years, distinct endocytic compartments, defined as subapical compartment or common endosome, were shown to play a prominent role in regulating membrane trafficking to and from polarized membrane domains. Sorting devices remain to be determined but likely include distinct rab proteins, and evidence is accumulating to indicate that signaling events may direct intracellular membrane transport, intimately involved in the biogenesis and maintenance of polarized membrane domains and hence the development of cell polarity.

Role of rab proteins in epithelial membrane traffic.

Abstract: Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such may define functional organelle identity Most rabs are ubiquitously expressed However, some rabs are preferentially expressed in epithelial cells where they appear intimately associated with the epithelial-specific transcytotic pathway and/or tight junctions This review discusses the role of rabs in epithelial membrane transport

Insulin-like growth factor-binding protein 6 inhibits survival and differentiation of rat oligodendrocyte precursor cells

Abstract: Insulin-like growth factor 1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and promotes myelination. We demonstrate that IGF-binding protein 6 (IGFBP-6) is expressed and localized to the Golgi complex in rat oligodendrocyte precursor (O2A) cells. IGFBP-6 mRNA showed a developmentally regulated expression pattern, displaying a transient decrease during early development, and enhanced levels upon cell maturation. IGFBP-6 mRNA expression could be reduced by addition of basic fibroblast growth factor and progesterone while estrogen increased IGFBP-6 mRNA. IGF-1, platelet-derived growth factor, and insulin had no effect. When added exogenously, IGFBP-6 reduced O2A cell survival in the absence of IGF-1 and inhibited IGF-1-stimulated survival in a partially IGF-1-dependent and partially IGF-1-independent fashion. In addition, IGFBP-6 reduced the IGF-stimulated expression of two myelin proteins, CNPase and MAG. Taken together, the data show that IGFBP-6 is a new negative effector of oligodendrocyte survival and differentiation.

Insulin-induced lipid binding to hemoglobin

Abstract: Under hypoglycemic conditions, concomitant hyperinsulinism causes an apparent modification of hemoglobin (Hb) which is manifested by its aggregation (Niketi} et al., Clin. Chim. Acta 197 (1991) 47). In the present work the causes and mechanisms underlying this Hb modification were studied. Hemoglobin isolated from normal erythrocytes incu- bated with insulin was analyzed by applying 31 P-spectrometry and lipid extraction and anal- ysis. To study the dynamics of the plasma membrane during hyperinsulinism, a fluorescent lipid-analog was applied. In the presence of insulin, phosphatidylserine (PS), phospha- tidylethanolamine (PE) and cholesterol were found to bind to Hb. Lipid binding resulted in Hb aggregation, a condition that can be reproduced when phospholipids are incubated with Hb in vitro. Using a fluorescent lipid-analog, it was also shown that exposing erythrocytes to supraphysiological concentrations of insulin in vitro resulted in the internalization of lipids. The results presented in this work may have relevance to cases of diabetes mellitus and hypoglycemia.

Lipoplex assembly visualized by atomic force microscopy

Abstract: Publisher Summary This chapter discusses the experimental approaches to deliver DNA into eukaryotic cells, a process also known as “transfection.” The purpose of such applications may range from substituting malfunctioning genes in cells to expressing novel genes for fundamental cell biological investigations. The development of an appropriate vehicle for the effective (intra-) cellular delivery of the gene(s) of interest has represented a major challenge. The chapter also focuses on the application of atomic force microscopy as a useful tool in the characterization of gene complexes composed of plasmid DNA and cationic lipids. Because of their positive charge, cationic lipids interact readily with the negatively charged DNA, resulting in the assembly of the so-called lipoplexes. When added to cells in culture, these complexes can be internalized, release their DNA, and, eventually, the genes encoded by the DNA are expressed. Hundreds of different cationic amphiphiles have been synthesized. Some important biophysical parameters of the cationic lipids that may drive distinct steps in the overall transfection process are gradually revealed, and many others remain to be determined. Atomic force microscopy (AFM) offers such possibilities. The application of this versatile technique in studies involving the characterization of the lipoplex assembly is described in the chapter.