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J Lippincott-Schwartz

Publications and source records attributed to J Lippincott-Schwartz.

At least 55 records · Page 3Linked to original sources
PubMed1997

ER-to-Golgi transport visualized in living cells.

Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.

Animals↗
PubMed1997

Insights into secretory and endocytic membrane traffic using green fluorescent protein chimeras.

Green fluorescent fusion proteins, which can be visualized in the unperturbed environment of a living cell, have become important reporter molecules for studying protein localization and trafficking within secretory and endocytic membranes of living cells. They have been used in a wide variety of applications, including time-lapse imaging, double-labeling and photobleach experiments. Results from such work are clarifying the steps involved in the formation, translocation and fusion of transport intermediates, are defining the roles for microtubules in membrane transport, and are providing insights into the mechanisms of protein retention and localization within organelles. In so doing, they have changed our thinking about the temporal and spatial relationships between subcellular membrane structures and the morphogenesis of secretory and endocytic organelles.

Animals↗
PubMed1996

Diffusional mobility of Golgi proteins in membranes of living cells.

The mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid is not yet understood. The diffusional mobilities of mammalian Golgi membrane proteins fused with green fluorescent protein from Aequorea victoria were measured in living HeLa cells with the fluorescence photobleaching recovery technique. The diffusion coefficients ranged from 3 x 10(-9) square centimeters per second to 5 x 10(-9) square centimeters per second, with greater than 90 percent of the chimeric proteins mobile. Extensive lateral diffusion of the chimeric proteins occurred between Golgi stacks. Thus, the chimeras diffuse rapidly and freely in Golgi membranes, which suggests that Golgi targeting and retention of these molecules does not depend on protein immobilization.

Aluminum Compounds↗
PubMed1996

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.

Animals↗
PubMed1995

Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia.

A fundamental characteristic of eukaryotic cells is the presence of membrane-bound compartments and membrane transport pathways in which the Golgi complex plays a central role in the selective processing, sorting, and secretion of proteins. The parasitic protozoan Giardia lamblia belongs to the earliest identified lineage among eukaryotes and therefore offers unique insight into the progression from primitive to more complex eukaryotic cells. Here, we report that Giardia trophozoites undergo a developmental induction of Golgi enzyme activities, which correlates with the appearance of a morphologically identifiable Golgi complex, as they differentiate to cysts. Prior to this induction, no morphologically or biochemically identifiable Golgi complex exists within nonencysting cells. Remarkably, protein secretion in both nonencysting and encysting trophozoites is inhibited by brefeldin A, and brefeldin A-sensitive membrane association of ADP-ribosylation factor and beta-COP is observed. These results suggest that the secretory machinery of Giardia resembles that of higher eukaryotes despite the absence of a Golgi complex in nonencysting trophozoites. These findings have implications both for defining the minimal machinery for protein secretion in eukaryotes and for examining the biogenesis of Golgi structure and function.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Organization of organelles and membrane traffic by microtubules.

Organelles of the central membrane system of higher eukaryotes have been shown to utilize microtubules both for maintenance of their characteristic spatial distributions and for efficient transport of their protein and lipid to diverse sites within the cell. Recent work addressing the mechanisms that underlie this organization provides new insights regarding the roles of microtubules and microtubule motors in influencing organelle dynamics and specific membrane traffic routes through the cytoplasm.

Animals↗
PubMed1995

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic.

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre-Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex.

Animals↗
PubMed1993

Membrane cycling between the ER and Golgi apparatus and its role in biosynthetic transport.

Membrane traffic between the ER and Golgi is now recognized as a carefully regulated process controlled by distinct anterograde (to the Golgi) and retrograde (to the ER) pathways. These pathways link two organelles with different morphologies, structures, and localizations within the cell. The ER, which is involved in multiple cellular functions including protein biosynthesis and folding, extends to the cell periphery and forms a dynamic tubule reticulum. By contrast, the Golgi apparatus, which functions in membrane sorting and recycling events, is localized at the center of the cell near the MTOC and is comprised of compact cisternal units. The required transport into the Golgi apparatus of newly synthesized proteins exported from the ER offers a twofold advantage to the cell. First, the rate of movement of membrane and protein through the biosynthetic pathway can be controlled by the selective use of a recycling pathway. Second, membrane moving through the biosynthetic pathway enters a structure specialized for sorting of membrane to different final destinations in the cell Control of biosynthetic transport within the ER/Golgi system involves the utilization of two alternative transport pathways: anterograde (ER to Golgi) and retrograde (Golgi to ER). These two pathways share a common regulatory system involving membrane assembly/disassembly of cytosolic coatomer proteins. Thus, conditions that favor irreversible coatomer binding (i.e., GTP gamma S) inhibit retrograde transport while producing anterograde transport intermediates. Conditions that prevent coatomer binding (i.e., BFA) inhibit anterograde transport and enhance retrograde transport. The underlying biochemical machinery that normally balances anterograde and retrograde membrane traffic between the ER and Golgi is only just beginning to be understood. Any model to explain this system, however, must account for the morphologic characteristics of the membranes involved. Whereas anterograde traffic involves discontinuous "coated" structures moving from peripheral sites in the ER toward the central Golgi, retrograde traffic utilizes continuous "noncoated" tubule structures that move from a central site (i.e., the CGN) to the peripheral ER (see Figure 3). Such a system maximizes volume transport (utilizing vacuolar structures) in the anterograde direction and membrane transport (utilizing tubules) in the retrograde direction. It is therefore ideal for sorting of bulk flow lumenal contents from recycling membrane early in the biosynthetic pathway.

Animals↗
PubMed1993

Bidirectional membrane traffic between the endoplasmic reticulum and Golgi apparatus.

Membrane traffic between the endoplasmic reticulum and Golgi apparatus is a highly regulated process that uses distinct anterograde and retrograde pathways. These pathways link two organelles that together function as a dynamic membrane system specialized for the biosynthesis and sorting of membrane to be used throughout the cell. The nature and underlying biochemical control of membrane transport along these pathways is thought to be tied to a common regulatory system involving assembly and disassembly of cytosolic proteins on membranes.

Journal Article↗
PubMed1991

Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein.

The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with the Golgi apparatus in an energy, GTP, and fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, the 110-kilodalton cytosolic coat protein beta-COP. Addition of beta gamma subunits of G proteins inhibited the association of both ARF and beta-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Thus, heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane.

ADP-Ribosylation Factors↗
PubMed1991

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.

Animals↗
PubMed1991

A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules.

Assembly of class I major histocompatibility complex (MHC) molecules involves the interaction of two distinct polypeptides (the heavy and light chains) with peptide antigen. Cell lines synthesizing both chains but expressing low levels of MHC class I molecules on their surface as a result of a failure in assembly and transport have been identified. We now report that although the apparent steady-state distribution in these cells of class I molecules is in the endoplasmic reticulum (ER), the molecules in fact are recycled between the ER and Golgi, rather than retained in the ER. This explains the failure of class I molecules to negotiate the secretory pathway. Class I molecules do not seem to be modified by Golgi enzymes, suggesting that the proteins do not reach the Golgi apparatus during recycling. But morphological and subcellular fractionation evidence indicates that they pass through the cis Golgi or a Golgi-associated organelle, which we postulate to be the recycling organelle. This compartment, which we call the 'cis-Golgi network', would thereby be a sorting organelle that selects proteins for return to the ER.

Animals↗
PubMed1991

Novel redistribution of an intracellular pool of CD45 accompanies T cell activation.

The major tyrosine phosphatase activity against angiotensin detected in membranes of the antigen-specific T cell hybridoma 2B4 is contained in the cytoplasmic tail of the CD45 molecule. When these cells are stimulated with either an antibody directed against the T cell antigen receptor or an activating anti-Thy-1 antibody, there is a rapid redistribution of CD45 in the cells. The redistribution can be observed in two ways: morphology and subcellular fractionation. Morphologic examination of resting cells reveals intense CD45 staining of the Golgi as well as surface staining. Upon activation the Golgi is rapidly cleared of CD45. This redistribution is specific for CD45 and is not observed for an intrinsic Golgi protein, mannosidase II, or a protein traversing the secretory pathway, the T cell receptor. In activated cells, in contrast to resting cells, approximately 30% of the total cellular CD45 is precipitated either at 280 x g or at 200,000 x g through a 2.2 M sucrose cushion after cell homogenization. This fraction is not accessible to cell surface labeling. CD45 redistribution does not require hydrolysis of phosphatidylinositides and cannot be reproduced by the addition of phorbol ester and calcium ionophore. It does require the presence of an intact functional T cell receptor on the cell surface. These studies suggest that the residence time of CD45 within an intracellular organelle can be acutely regulated by a signal mediated via the T cell receptor. This regulation may control access of this phosphatase to critical substrates.

Animals↗
PubMed1991

Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae.

We report that brefeldin A prevents the assembly of non-clathrin-coated vesicles from Golgi cisternae in a cell-free system. This finding provides a simple molecular explanation for the primary effect of this remarkable compound in blocking constitutive secretion. We further report that when coated vesicle assembly is blocked, extensive tubule networks form that connect previously separate cisternae and stacks into a single topological unit, allowing the intermixing of contents of Golgi cisternae, presumably by lateral diffusion. Formation of the tubule networks requires ATP, cytosol, and the general fusion protein NSF. Tubule networks may be related to the membrane tubules mediating retrograde transport in vivo.

Animals↗
PubMed1991

Degradation of proteins within the endoplasmic reticulum.

Certain newly synthesized proteins within the endoplasmic reticulum undergo rapid turnover by a non-lysosomal proteolytic pathway. Biochemical and morphological evidence has suggested that these proteins never leave the endoplasmic reticulum before they are degraded. The mechanism(s) for the selective targeting of proteins for degradation within the endoplasmic reticulum is still not understood, but appears to rely on specific structural determinants on the protein substrates. Important cellular functions are likely to be served by this endoplasmic reticulum degradative system, including disposal of abnormal proteins and the selective turnover of metabolically regulated proteins.

Animals↗
PubMed1991

Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.

4-Chloro-7-nitrobenzofurazan↗