Impairing Actin Filament or Syndapin Functions Promotes Accumulation of Clathrin-coated Vesicles at the Apical Plasma Membrane of Acinar Epithelial Cells

Costa, Silvia R. da; Sou, Eunbyul; Xie, Jiansong; Yarber, Francie A.; Okamoto, Curtis T.; Pidgeon, Michael; Kessels, Michael M.; Mircheff, Austin K.; Schechter, Joel; Qualmann, Britta; Hamm‐Alvarez, Sarah F.

doi:10.1091/mbc.e03-05-0315

Cited by 46 publications

(51 citation statements)

References 45 publications

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“…The entire cell surface became highly irregular, presumably because of the normally stabilizing, cortical actin cytoskeleton was destroyed, so that a clear distinction between protrusions and bulk membrane was not possible (Figure 7, B and C). In agreement with other studies of the relation between the actin cytoskeleton and the clathrinmediated endocytosis (Gaidarov et al, 1999;Cao et al, 2003;da Costa et al, 2003), the number of clathrin-coated pits increased considerable. Most importantly, immunogold labeling did not reveal any ErbB2 in the coated pits ( Figure 7, B and C).…”

supporting

confidence: 92%

Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

Hommelgaard

Lerdrup

Deurs

2004

MBoC

187

201

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In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance. INTRODUCTIONErbB2, a member of the epidermal growth factor (EGF) receptor (EGFR) family, has no specific ligand, but it is the main heterodimerization partner for the other family members (Sliwkowski et al., 1994;Yarden, 2001;Yarden and Sliwkowski, 2001). The dimerization is mediated by a unique dimerization arm localized to the extracellular (Nterminal) portion of the receptor. Whereas exposure of this arm normally requires ligand binding and reorganization of the exterior portion of ErbB receptors, it is constitutively exposed in ErbB2 (Burgess et al., 2003). Moreover, in contrast to the EGFR, which is rapidly down-regulated upon ligand stimulation (Sorkin and Von Zastrow, 2002), ErbB2 avoids delivery to lysosomes and subsequent proteolysis and has a long half-life at the plasma membrane (Gilboa et al., 1995;Baulida et al., 1996;Baulida and Carpenter, 1997;Waterman et al., 1998;Wang et al., 1999). ErbB2 expression is up-regulated in various cancers. For instance, 20 -30% of breast cancers show strong overexpression, and this correlates with poor prognosis (Slamon et al., 1989;Reese and Slamon, 1997;Eccles, 2001). The oncogenic potential of ErbB2 may in part be explained by its capability to avoid down-regulation together with its pronounced engagement in formation of heterodimers. When overexpressed, ErbB2 causes the other family members to escape the lysosomal pathway upon ligand binding and receptor activation (Yarden, 2001). However, the reason why ErbB2 escapes the lysosomal pathway and remains at the plasma membrane has remained elusive. Basically, two mechanisms could lead to retention of ErbB2 at the plasma membrane: either E...

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supporting

confidence: 92%

Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

Hommelgaard

Lerdrup

Deurs

2004

MBoC

187

201

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“…Overexpression of PACSIN isoforms in NIH 3T3 fibroblasts inhibits transferrin uptake [11]. Introduction of the SH3 domain of PACSIN results in filamentous actin accumulation and impairment in endocytosis, as assayed by apical plasma membrane accumulation of α-adaptin and clathrin [5]. Here we show that PACSIN3 participates in the trafficking of GLUT1, but not GLUT4, in unstimulated 3T3-L1 adipocytes.…”

mentioning

confidence: 56%

“…Recent studies have identified adaptor proteins that do coordinate membrane trafficking events with alterations in the cytoskeleton. Major examples are the amphiphysin, cortactin, endophilin, intersectin and syndapin/PACSIN families [4][5][6][7]. These proteins contain multiple protein-protein interaction domains, which allow them to *Address correspondence to William Roach at present address: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844. william.roach@dartmouth.edu.…”

Section: Introductionmentioning

confidence: 99%

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

Roach¹,

Plomann²

2007

Biochemical and Biophysical Research Communications

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PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes. Keywordsadipocyte; endocytosis; GLUT1; GLUT4; PACSIN3; membrane trafficking; syndapin The need to transport glucose from outside to inside the cell is a fundamental feature of cellular survival and growth. In mammals glucose transport of the facilitated diffusion type is mediated by a group of transporters known as the GLUT family (SLC2A gene symbol). The GLUT's comprise a 13-member family of 12-trans-membrane spanning 50-60 kD proteins [1]. Among these, GLUT1 and GLUT4 are of central relevance to peripheral glucose disposal and have been extensively studied. Nevertheless, the mechanisms involved in controlling the trafficking of GLUT1 and GLUT4 to and from the plasma membrane, and the differences between the trafficking of the two GLUT's, are not completely understood [2,3]. It is possible that proteins involved in integrating cytoskeletal rearrangements with membrane trafficking participate in these mechanisms. Recent studies have identified adaptor proteins that do coordinate membrane trafficking events with alterations in the cytoskeleton. Major examples are the amphiphysin, cortactin, endophilin, intersectin and syndapin/PACSIN families [4][5][6][7]. These proteins contain multiple protein-protein interaction domains, which allow them to *Address correspondence to William Roach at present address: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844. william.roach@dartmouth.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In thi...

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“…23 Digital image analysis and evaluation of A/D rab3D labeling ratios in multiple planes from z-stacks acquired from acini was as described previously. 54 Serial sections from confocal fluorescence microscopy images of nontransduced and Ad-LacZ-transduced acini of comparable size and thickness labeled to detect actin filaments and rab3D were acquired at equal gain and contrast intensities, pinhole size, image section thickness (0.6 mm) and zoom ( Â 2 zoom magnification, Â 60 objective). Initial gain and contrast levels were established using both samples to ensure that fluorescence levels were not saturated.…”

Section: Confocal Fluorescence Microscopymentioning

confidence: 99%

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

et al. 2004

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Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in 480% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UVinactivated Ad, carbachol-stimulated release of bulk protein and b-hexosaminidase were significantly (Pp0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

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Impairing Actin Filament or Syndapin Functions Promotes Accumulation of Clathrin-coated Vesicles at the Apical Plasma Membrane of Acinar Epithelial Cells

Cited by 46 publications

References 45 publications

Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

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