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Polarization of Myelinating Schwann Cell Surface Membranes: Role of Microtubules and the Trans-Golgi Network

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Polarization of Myelinating Schwann Cell Surface Membranes: Role of Microtubules and the Trans-Golgi Network The Journal of Neuroscience, March 1995, 15(3): 1797-1807 Polarization of Myelinating Schwann Cell Surface Membranes: Role of Microtubules and the Trans-Golgi Network Bruce D. Trapp,’ Grahame J. Kidd, 2,a Peter Hauer,* Eileen Mulrenin,’ Carol A. Haney,* and S. Brian Andrews3 ‘Department of Neurosciences, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, *Department of Neurology, The Johns Hopkins University School of Medicine, Neuromuscular Division, Baltimore, Maryland 21287- 6965, and 3Laboratory of Neurobiology, NINDS, National Institutes of Health, Bethesda, Maryland 20892 Schwann cells polarize their surface membranes into sev- The surface membranes of most eukaryotic cells are divided into eral biochemically and ultrastructurally discrete regions of specialized regions that have different molecular compositions the myelin internode. To form these membrane domains, and functions. Plasma membranes in cells that extend long pro- Schwann cells must sort, transport, and target membrane cesses can be very complex. The surface membranes of neurons, proteins appropriately. In this study, microtubule disas- for example, can be divided into two biochemically and func- sembly, confocal microscopy, and electron microscopic tionally distinct domains, axons and dendrites, both of which immunocytochemistry were used to investigate mecha- can be further divided into multiple subdomains. Polarization of nisms involved in targeting P, protein (P,), the myelin-as- cell surfaces into discrete regions is a complex process that must sociated glycoprotein (MAG), and laminin to different plas- result in site-specific delivery of membrane components. Protein ma membrane domains in myelinating Schwann cells from sorting, transport, integration, and stabilization are just a few of 35d-old rat sciatic nerve. After microtubule disassembly the mechanisms whereby cells establish unique plasma mem- by colchicine, all three proteins accumulated in Schwann brane domains. cell perinuclear cytoplasm, indicating that microtubules Much of what is known about mechanisms of protein sorting are necessary for their transport. The distributions of Golgi and targeting has been obtained from in vitro studies of epithelial membranes, endoplasmic reticulum, and intermediate fila- cells that polarize their surfaces into apical and basolateral do- ments were also altered by colchicine treatment. Electron mains (Rodriguez-Boulan and Nelson, 1989; Simons and Wan- microscopic immunocytochemical studies indicated that P, dinger-Ness, 1990; Mostov et al., 1992). Epithelial cells target and MAG are sorted into separate carrier vesicles as they proteins from Golgi membranes to their correct surface domains exit the trans-Golgi network. Following microtubule disas- by two pathways. In one pathway, segregation of newly synthe- sembly, P,-rich carrier vesicles fused and formed myelin- sized glycoproteins occurs in the trans-Golgi network, where like membrane whorls, whereas MAG-rich carrier vesicles basolateral and apical proteins are sorted into separate carrier fused and formed mesaxon-like membrane whorls. Micro- vesicles that are then targeted directly to the apical or basolateral tubule disassembly did not result in mistargeting of either plasma membrane domains (Rindler et al., 1984; Griffiths and P, or MAG to surface membranes. These results indicate Simons, 1986; Rodriguez-Boulan and Nelson, 1989). The sec- that following sorting in the trans-Golgi network, certain ond pathway transports both apical and basolateral proteins to carrier vesicles are transported along the myelin internode the basolateral surface. Apical proteins are then removed from on microtubules; however, microtubules do not appear to the basolateral membrane by endocytosis and relocated to the target these vesicles selectively to specific sites. The tar- apical surface (Hubbard and Stieger, 1989). Different epithelial MAG-, and laminin-rich carrier vesicles to geting of PO-, cells use these two transport pathways to different degrees (Rin- specific sites most likely occurs by ligand receptor binding dler et al., 1987; Achier et al., 1989; Eilers et al., 1989; LeBivic mechanisms that permit fusion of carrier vesicles only with et al., 1990; Matter et al., 1990). In many cases, specialized the appropriate target membrane. microtubule (MT) organizations facilitate the delivery of carrier [Key words; protein sorting, protein targeting, PO protein, vesicles to specific membrane domains (Hugon et al., 1987; myelinsssociated glycoprotein, microtubules, myelina- Achier et al., 1989; Bre et al., 1990; Gilbert et al., 1991). tion] Schwann cells, the myelin-forming cell in the peripheral nervous system (PNS), polarize their surfaces into multiple Received May 20, 1994; revised Aug. 15, 1994; accepted Aug. 22, 1994. membrane domains. Three glycoproteins-P, protein (P,,), the We thank Dr. Pamela Talalay for helpful comments, Dr. Chris Linington for myelin-associated glycoprotein (MAG), and laminin-are syn- P, monoclonal antibodies, Rod Graham for editing and preparing the manu- thesized in rough endoplasmic reticulum (RER), processed script, and Diana Curley for technical assistance. This work was supported by grants NS 22849 and NS 29818 from the National Institute of Neurological through the Golgi, and then targeted to different Schwann cell Disorders and Stroke. Dr. Kidd is a postdoctoral fellow of the National Multiple surface membranes: P, to compact myelin (Trapp et al., 1981), Sclerosis Society. Correspondence should be addressed to Dr. Bruce Trapp, Department of MAG to noncompact myelin (Trapp and Quarles, 1982), and Neurosciences-NC30, The Cleveland Clinic Foundation, 9500 Euclid Avenue, laminin to the plasma membrane (Cornbrooks et al., 1983). My- Cleveland, OH 44195. elin basic protein (MBP) is targeted to compact myelin by trans- ‘Present address: Department of Biochemistry, University of Queensland, St. Lucia, Queensland, Australia 4067. location of its mRNA (Colman et al., 1982; Trapp et al., 1987). Copyright 0 I995 Society for Neuroscience 0270-6474/95/151797-l 1$05.00/O The outer perimeter of the myelin internode consists of cyto- 1798 Trapp et al. * Protein Targeting in Myelinating Schwann Cells plasmic-rich and cytoplasmic-free regions (Mugnaini et al., specimen stubs, and frozen in liquid nitrogen. Immunogold staining was 1977; Trapp et al., 198913). The cytoplasmic-rich regions form performed by previously described modifications (Trapp et al., 1988a, 1989a,b) of standard immunogold procedures (Slot and Geuze, 1984; channels that extend along the entire length of the internode. Tokuyasu, 1986). In brief, ultrathin cryosections (- 120 nm thick) were Proteins, lipids, and RNA are transported from the perinuclear cut on glass knives in a ultracryomicrotome (Ultracut FC4; Reichert cytoplasm to points along the internode within these channels Scientific Instruments) maintained at - 110°C. The sections were placed (Gould and Mattingly, 1990). Microtubules are abundant in these on carbon- and Formvar-coated grids, immunostained, and then placed on 2.5% glutaraldehyde and PBS for 15 min and rinsed. The sections channels (Peters et al., 1991; Kidd et al., 1994) and they prob- were subsequently stained with uranyl acetate and embedded in 1% ably help transport myelin proteins along the myelin internode. methyl cellulose containing 3% uranyl acetate. Immunostained cry- The present study investigates how P,, MAG, and laminin are osections were examined in a Hitachi H-600 electron microscope. sorted, transported, and targeted to different membrane domains. By using colchicine to reversibly depolymerize Schwann cell Results MTs in viva, this study demonstrates that P,, MAG, and laminin The present study depends on MT disassembly, a strategy that are sorted into separate carrier vesicles as they exit the trans- has been instrumental in delineating the function of MTs in a Golgi network. These vesicles are transported along the myelin variety of cell types (Rogalski and Singer, 1984; Rindler et al., internode on MTs, and then inserted directly into the appropriate 1987; Achier et al., 1989; Gilbert et al., 199 1). Microtubules can surface membrane domain. Our data also demonstrate that MTs be disassembled by a variety of methods, including exposure to maintain the distribution of Golgi membranes as well as net- depolymerizing drugs (colchicine, colcemid, nocodazole), low works of smooth endoplasmic reticulum (SER) and intermediate temperature, and hypertonic shock. Nocodazole is currently the filaments that extend along the outer perimeter of the myelin most widely used drug for inducing MT disassembly in vitro, internode. but it did not penetrate into sciatic nerves when applied by cuff. In contrast, consistent reversible MT disassembly was produced Materials and Methods by soaking rayon wool cuffs with 20 ~1 of 5 mM colchicine and Colchicine administration placing them around the sciatic nerve for 2 hr. Colchicine con- Sprague-Dawley rats (35 d old) were anesthetized and the left sciatic centrations higher than 5 mM caused Wallerian degeneration. nerve was immobilized in the midthigh. Two milligrams of rayon wool The appropriate colchicine concentration for producing revers- (obtained from sterile gauze pads) soaked in 20 ~1 of colchicine was ible MT disassembly can vary, however, depending on the sup- placed around the nerve. Care was taken not to constrict the nerve at either end of the rayon wool cuff. After 2 hr, the cuff was removed and plier and the batch of colchicine. the animal was allowed
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