D. Sheff - Academia.edu (original) (raw)

Papers by D. Sheff

The Journal of cell biology, Jan 4, 2002

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pas... more In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Ra...

Molecular biology of the cell, 2009

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and l... more Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with "leaky" nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for...

The Journal of Cell Biology, 2006

The Journal of Cell Biology, 1997

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are function... more Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit ⑀ -COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable ⑀ -COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type ⑀ -COP cDNA demonstrating that they directly or indirectly reflected the ⑀ -COP defect. Taken together, the results suggest that ⑀ -COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

The Journal of Cell Biology, 1999

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear ... more Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also Elizabeth A. Daro's current address is Immunex, 51 University St., Seattle, WA 98101.

Proceedings of the National Academy of Sciences, 1990

The recent recognition of the eosinophilia-myalgia syndrome (EMS) associated with the ingestion o... more The recent recognition of the eosinophilia-myalgia syndrome (EMS) associated with the ingestion of L-tryptophan prompted an analysis of the peripheral blood eosinophil phenotypes and of the serum eosinophil hematopoietins in this disorder. Five patients with an illness characterized by the abrupt onset of aching skeletal muscles, edema, thickening and induration of the skin, and marked blood eosinophilia associated with L-tryptophan ingestion provided eosinophils, serum, or both, for evaluation. Gradient sedimentation density analysis of the peripheral blood eosinophils from four of these patients revealed that 43 +/- 13% (mean +/- SEM) of the cells had converted to the abnormal (hypodense) sedimenting phenotype. When normodense eosinophils from the reference donors were cultured for 3 days in medium supplemented with increasing concentrations of serum from the patients with EMS, their viability increased in a dose-dependent manner to 45%, which was significantly augmented over the effect of normal serum. This eosinophil viability-sustaining activity was inhibited by 76 +/- 7% (mean +/- SEM; n = 3) by the addition of anti-interleukin 5 (IL-5) but not by neutralizing antibodies monospecific for either granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3. IL-5, an eosinophilopoietic factor, converts normodense peripheral blood eosinophils in vitro to a hypodense sedimenting form with extended viability and augmented biologic responses to activating stimuli. Thus, the presence of IL-5 in the sera of patients with EMS may contribute to the development and maintenance of the eosinophilia and may regulate the conversion of the peripheral blood eosinophils to the hypodense phenotype with augmented pathobiologic potential.

Proceedings of the National Academy of Sciences, 2011

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its lo... more Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown.

Nature, 2002

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The fi... more Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Molecular Biology of the Cell, 2008

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basol... more Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the 1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

Journal of Experimental Medicine, 1998

Cells from the bone marrow can present peptides that are derived from tumors, transplants, and se... more Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-A b MHC class II presenting a peptide derived from I-E ␣ . When immature DCs from I-A b mice were cultured for 5-20 h with activated I-E ϩ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DR ␣ includes the same peptide sequence as mouse I-E ␣ . Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-Ebearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self.

Journal of Cell Science, 2009

Journal of Biological Chemistry, 1996

The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membra... more The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membrane-associated coats thought to mediate vesicular transport between the endoplasmic reticulum and the Golgi complex, between adjacent Golgi cisternae, and possibly in the endocytic pathway. We investigated whether this heterogeneity in the sites of COPI action might be reflected in biochemical heterogeneity of one or more COPI subunits. A simplified method was devised to purify the cytosolic COPI precursor complex, coatomer, from rat liver cytosol. The individual subunits were analyzed by high resolution two dimensional gel electrophoresis and mass spectroscopic analysis of tryptic peptides. Considerable charge heterogeneity was observed, particularly for the beta-COP and delta-COP subunits. The multiple species detected, however, did not appear to reflect the presence of distinct translation products but rather a significant degree of protein phosphorylation. The observed pI of beta-COP was sensitive to alkaline phosphatase digestion. Moreover, isolation of coatomer from metabolically labeled tissue culture cells demonstrated directly that both beta-COP and delta-COP, but no other coatomer subunits, were serine-phosphorylated. COPI phosphorylation may regulate coatomer assembly, membrane recruitment, or the specificity of coatomer-organelle interaction.

Journal of Biological Chemistry, 2012

Background: GABA B R signaling blocks neuronal firing ensuring appropriate cerebellar cortex outp... more Background: GABA B R signaling blocks neuronal firing ensuring appropriate cerebellar cortex output. Results: Loss of RGS6 results in ataxia rescued by a GABA B R antagonist and enhanced GABA B R-GIRK current in neurons. Conclusion: RGS6 is an essential component of GABA signaling in cerebellum and required for motor coordination. Significance: RGS6 dysregulation could result in cerebellar ataxia, and thus, it might represent a novel target for pharmacological intervention. . 4 The abbreviations used are: CGN, cerebellar granule neuron; DEP/DHEX, Dishevelled, Egl-10 and Pleckstrin homology domain; GABA, ␥-aminobutyric acid; GABA B R, metabotropic GABA receptor; GAP, GTPase-accelerating protein; GGL, G␥ subunit-like domain; GIRK, G protein-activated inwardly rectifying potassium channel; GPCR, G protein-coupled receptor; KO, knockout; RGS, regulator of G protein signaling; RGS6, regulator of G protein signaling 6; R7BP, R7 family RGS binding protein. Downloaded from FIGURE 8. Loss of RGS6 potentiates GABA B R-GIRK signaling in isolated CGNs. A, representative baclofen (200 nM)-induced whole cell GIRK current recordings in isolated CGNs from WT and RGS6 Ϫ/Ϫ mice. GIRK currents from RGS6 Ϫ/Ϫ mice exhibit (B) a significant delay in the half-maximal deactivation but not activation time constant ( 1/2 ) without any significant changes in the extent of desensitization (C) and current density (D) (WT, n ϭ 8; RGS6 Ϫ/Ϫ , n ϭ 10). Data are presented as mean ϮS.E.; *, p Ͻ 0.05 versus WT.

Arthritis & Rheumatism, 1997

To examine the sensitivity and positive predictive value of Medicare physician claims for select ... more To examine the sensitivity and positive predictive value of Medicare physician claims for select rheumatic conditions managed in rheumatology specialty practices. Eight rheumatologists in 3 states abstracted 378 patient office records to obtain information on diagnosis and office procedures. The Medicare Part B physician claims for these patient visits were obtained from the Health Care Financing Administration. The sensitivity of the claims data for a specific diagnosis was calculated as the proportion of all patients whose office records for a particular visit documented that diagnosis and who also had physician claims for that visit which identified that diagnosis. The positive predictive value was evaluated in a separate sample of 331 patient visits identified in Medicare physician claims. The positive predictive value of the claims data for a specific diagnosis was calculated as the proportion of patients with that diagnosis coded in the claims for a particular visit who also had the diagnosis documented in the medical record for that visit. Ninety percent of abstracted office medical records were matched successfully with Medicare physician claims. The sensitivity of the Medicare physician claims was 0.90 (95% confidence interval [CI] 0.85-0.95) for rheumatoid arthritis (RA), 0.85 (95% CI 0.73-0.97) for systemic lupus erythematosus (SLE), and 0.85 (95% CI 0.78-1.0) for aspiration or injection procedures. The sensitivity for osteoarthritis (OA) of the hip or knee was &amp;lt; or = 0.50 if 5-digit codes specifying anatomic site were required. The sensitivity for fibromyalgia (FM) was 0.48 (95% CI 0.28-0.68). The positive predictive values were at least 0.90 for RA, SLE, and aspiration or injection procedures. Positive predictive values for FM and the 5-digit site-specific codes for OA of the knee were 0.83 (95% CI 0.66-1.0) and 0.88 (95% CI 0.75-1.0), respectively, while the positive predictive value of the 5-digit site-specific codes for OA of the hip was zero (95% CI 0-0.26). The positive predictive value of OA at any site was 0.83 (95% CI 0.76-0.90). In specialty practice, Medicare physician claims had high sensitivity and positive predictive value for RA, SLE, OA without specification of anatomic site, and injection or aspiration procedures. The claims had lower sensitivity and predictive value for FM and for OA of the hip. The accuracy of Medicare physician claims for other conditions and in the primary care setting requires further investigation.

Molecular Biology of the Cell, 2011

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for re... more Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identifi ed, questions remain concerning how specifi c functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specifi c composition of TJs.