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Biomedical subjects

David J Stephens

Publications and source records attributed to David J Stephens.

At least 19 recordsLinked to original sources
PubMed2006

Nordihydroguaiaretic acid affects multiple dynein-dynactin functions in interphase and mitotic cells.

Nordihydroguaiaretic acid (NDGA), a well known lipoxygenase inhibitor, actually has pleiotropic effects on cells, which include cell proliferation, apoptosis, differentiation, and chemotaxis. We and others have shown previously that this compound causes Golgi disassembly by an unknown mechanism. In this study, we show that, in parallel with Golgi disassembly, NDGA induces the accumulation of the microtubule minus-end-directed motor dynein-dynactin complex at the centrosome, where microtubules minus-ends lie. Concomitant with this accumulation, dynein-dynactin-interacting proteins, such as ZW10 and EB1, were also redistributed to the centrosomal region. In cells where microtubules were depolymerized by nocodazole, NDGA promoted the formation of filaments consisting of dynein-dynactin and its interacting proteins, suggesting that it stimulates the association of these proteins in an ordered, not random, manner. Loss of dynactin function abolished not only NDGA-induced redistribution in intact cells but also filament formation in nocodazole-treated cells. The latter finding implies that dynactin is a key molecule for the association between dynein-dynactin and its interacting proteins. In mitotic cells, NDGA induced robust accumulation of dyneindynactin and its interacting proteins at the spindle poles. These results taken together suggest that NDGA perturbs membrane traffic by affecting the function of the microtubule motor dynein-dynactin complex and its auxiliary proteins. To our knowledge, NDGA is the first case of a reagent that can modulate dynein-dynactin-related processes.

Centrosome↗
PubMed2006

Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells.

The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.

Cell Line↗
PubMed2006

Microtubule plus-end loading of p150(Glued) is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells.

Microtubule dynamics and function are regulated, at least in part, by a family of proteins that localize to microtubule plus-ends, and include EB1, CLIP-170 and the dynactin component p150(Glued). Plus-end pools of these proteins, notably dynactin, have been invoked in a number of ;search-and-capture' mechanisms, including the attachment of microtubules to kinetochores during mitosis and to endomembranes prior to the initiation of intracellular transport. Here we show that, in mammalian cells, EB1 is required for the plus-end localization of CLIP-170, and that this is in turn required to localize p150(Glued) to plus-ends. Specific depletion of CLIP-170 results in defects in microtubule dynamics, cell polarization in response to scratch wounding and a loss of p150(Glued) from plus ends. By contrast, removal of p150(Glued) from plus-ends by depletion of either EB1 or CLIP-170 caused no defects in the localization of intracellular organelles, the dynamics of ER-to-Golgi transport, the efficiency of transferrin uptake or the motility of early endosomes or lysosomes. In addition to labelling microtubule plus-ends, we show that GFP-p150(Glued) becomes incorporated into the dynactin complex and labels small, highly dynamic, punctate structures that move along microtubules. A subset of these structures colocalizes with ER-Golgi transport intermediates. Together, these data show that the function of CLIP-170 and p150(Glued) in membrane trafficking is not associated with their plus-end localization.

Biological Transport↗
PubMed2006

Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor.

The type II alpha isoform of phosphatidylinositol 4-kinase has recently been shown to function in the recruitment of adaptor protein-1 complexes to the trans-Golgi network. Here we show that phosphatidylinositol 4-kinase IIalpha is also a component of highly dynamic membranes of the endosomal system where it colocalises with protein markers of the late endosome and with endocytosed epidermal growth factor. When phosphatidylinositol 4-kinase IIalpha activity was inhibited in vivo using the monoclonal antibody 4C5G or by depression of endogenous phosphatidylinositol 4-kinase IIalpha protein levels using RNA interference, ligand-bound epidermal growth factor receptor failed to traffic to late endosomes and instead accumulated in vesicles in a sub-plasma membrane compartment. Furthermore, lysosomal degradation of activated epidermal growth factor receptor was dramatically impaired in small inhibitory RNA-treated cells. We demonstrate that phosphatidylinositol 4-kinase IIalpha is necessary for the correct endocytic traffic and downregulation of activated epidermal growth factor receptor.

Antibodies, Monoclonal↗
PubMed2006

Secretory cargo regulates the turnover of COPII subunits at single ER exit sites.

The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes . Both stimulate the GTPase activity of Sar1p , which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.

Animals↗
PubMed2005

PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport.

The export of secretory cargo from the endoplasmic reticulum is mediated by the COPII complex. In common with other aspects of intracellular transport, this step is regulated by protein kinase signalling. Recruitment of the COPII complex to the membrane is known to require ATP and to be blocked by the protein kinase inhibitor H-89. The identity of the specific protein kinase or kinases involved remains equivocal. Here we show that the Sec23p subunit of COPII interacts with PCTAIRE protein kinases. This interaction is shown using two-hybrid screening, direct binding and immunoprecipitation. Inhibition of PCTAIRE kinase activity by expression of a kinase-dead mutant, or specific depletion of PCTAIRE using RNAi, leads to defects in early secretory pathway function including cargo transport, as well as vesicular-tubular transport carrier (VTC) and Golgi localization. These data show a role for PCTAIRE protein kinase function in membrane traffic through the early secretory pathway.

Animals↗
PubMed2005

ER-to-Golgi transport: form and formation of vesicular and tubular carriers.

The transport of proteins and lipids between the endoplasmic reticulum and Golgi apparatus is initiated by the collection of secretory cargo from within the lumen of the endoplasmic reticulum. Subsequently, transport carriers are formed that bud from this membrane and are transported to, and subsequently merge with, the Golgi. The principle driving force behind the budding process is the multi-subunit coat protein complex, COPII. A considerable amount of information is now available regarding the molecular mechanisms by which COPII components operate together to drive cargo selection and transport carrier formation. In contrast, the precise nature of the transport carriers formed is still a matter of considerable debate. Vesicular and tubular carriers have been characterized that are, or in other cases are not, coated with the COPII complex. Here, we seek to integrate much of the data surrounding this topic and try to understand the mechanisms by which vesicular and/or tubular carriers might be generated.

Animals↗
PubMed2005

Intracellular trafficking pathways and drug delivery: fluorescence imaging of living and fixed cells.

Cellular processes depend on the fidelity of intracellular membrane traffic. Lipids, proteins, receptor ligands and solute molecules are trafficked to distinct compartments within the cell through both the biosynthetic and endocytic pathways. An appreciation of these pathways is vital for a complete understanding of intracellular drug delivery. Recent advances in fluorescence imaging have facilitated the analysis of these pathways in great detail. It is now possible to gain insight into the real-time dynamics of cellular components and macromolecular pharmacological agents as they are delivered into and traffic within single cells. Here, we discuss the analysis of intracellular drug delivery from the perspective of fluorescence imaging of both living and fixed cells. This review aims to cover trafficking pathways, markers for subcellular compartments, fluorescent labels for intracellular structures and pharmacological agents and relevant recent developments in imaging technology. In particular, we shall focus on the application of live cell imaging to the study of endocytic drug delivery.

Bacterial Toxins↗
PubMed2005

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells.

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.

Animals↗
PubMed2005

Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells.

The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy. The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 258C) in the absence of pH gradient, ATP or other energy source. The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected. In contrast, when the peptide was attached to an IAF-labelled 25 kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells. The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.

ADP Ribose Transferases↗
PubMed2005

Measuring the induction or inhibition of apoptosis by HPV proteins.

Many viral proteins influence the cellular pathways that control cell proliferation and cell death. Some viral proteins trigger apoptotic cell death, and this may be important in host defense and viral spread. In other cases, viral proteins inhibit apoptosis. In this chapter, we will describe some of the methods that can be used to investigate the induction or inhibition of apoptosis by papillomavirus proteins.

Animals↗
PubMed2004

Coupling of ER exit to microtubules through direct interaction of COPII with dynactin.

Transport of proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the sequential action of two coat complexes: COPII concentrates cargo for secretion at ER export sites, then COPI is subsequently recruited to nascent carriers and retrieves recycling proteins back to the ER. These carriers then move towards the Golgi along microtubules, driven by the dynein/dynactin complexes. Here we show that the Sec23p component of the COPII complex directly interacts with the dynactin complex through the carboxy-terminal cargo-binding domain of p150(Glued). Functional assays, including measurements of the rate of recycling of COPII on the ER membrane and quantitative analyses of secretion, indicate that this interaction underlies functional coupling of ER export to microtubules. Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus.

Animals↗
PubMed2004

Differential effects of a GTP-restricted mutant of Sar1p on segregation of cargo during export from the endoplasmic reticulum.

Export of cargo from the endoplasmic reticulum (ER) is the first membrane trafficking step in the secretory pathway. To date, all cargo proteins appear to use a common set of machinery for the initial stages of export, namely the COPII coat complex. Recent data from both yeast and mammalian systems have emerged suggesting that specific cargoes could be sorted from one another at the point of exit from the endoplasmic reticulum or immediately afterwards. Here, we have examined the mechanisms used for export of different types of cargo molecule from the endoplasmic reticulum. All cargoes examined utilise the COPII machinery, but specific differences are seen in the accumulation of cargo into ER-derived pre-budding complexes following expression of a GTP-restricted mutant of the Sar1p GTPase. Glycosylphosphatidylinositol (GPI)-anchored GFP is seen to be restricted to the ER under these conditions whereas other cargoes, including ts045-G and lumFP accumulate in pre-budding complexes. Following exit, GPI-FP, lumFP and ts045-G-FP all travel to the Golgi in the same vesicular tubular clusters (VTCs). These data show a differential requirement for efficient GTP hydrolysis by the Sar1p GTPase in export of cargo from the ER.

Endoplasmic Reticulum↗
PubMed2004

Biogenesis of ER-to-Golgi transport carriers: complex roles of COPII in ER export.

It is widely believed that membrane traffic occurs by vesicular transport between successive compartments of the secretory pathway. Coat complexes function to collect cargo from donor membranes and deform them to generate transport vesicles with a diameter of 60-80 nm. Recent data argue in favour of a new model for export of secretory cargo from the endoplasmic reticulum, in which tubular extensions are protruded and subsequently matured into independent ER-to-Golgi transport carriers. Here, we examine the evidence for this controversial hypothesis.

Animals↗
PubMed2004

The intracellular transport of chylomicrons requires the small GTPase, Sar1b.

PURPOSE OF REVIEW: The transport of lipoproteins through the secretory pathways of enterocytes and hepatocytes is pivotal for whole-body lipid homeostasis. This review focuses on the assembly and structural evolution of COPII (coat protein) transport carriers that are essential for the transport of chylomicrons from the endoplasmic reticulum to the Golgi apparatus. RECENT FINDINGS: The assembly of endoplasmic reticulum to Golgi transport carriers commences with the coating of specific areas of the endoplasmic reticulum membrane with Sar1-GTP and the Sec23/24 heterodimer. An important advance has been the crystallographic analysis of the Sar1-Sec23/24 complex. The proteins form a bow-tie shaped structure, with a concave face that seems to match the curvature of transport carriers. Mammalian cells produce two isoforms of Sar1, designated Sar1a and Sar1b, both of which are expressed in enterocytes. Sar1b is defective in chylomicron retention disease and Anderson disease, two rare recessive disorders characterized by severe fat malabsorption and a failure to thrive in infancy. Patients with chylomicron retention disease and Anderson disease selectively retain chylomicron-like particles within membrane-bound compartments. By analogy with procollagen, chylomicrons may drive the formation of endoplasmic reticulum to Golgi transport carriers from endoplasmic reticulum sites close to, but separate from, domains of the endoplasmic reticulum coated with Sar1-Sec23/24. The COPII machinery also mediates the transport of VLDL to the Golgi. SUMMARY: New insights into the role of the COPII machinery in the intracellular transport of cargo, including chylomicrons and VLDL, may suggest new drug targets for ameliorating the lipid abnormalities of the metabolic syndrome.

Amino Acid Sequence↗
PubMed2003

Light microscopy techniques for live cell imaging.

Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. For anyone new to this area, however, it can be daunting to decide which techniques or equipment to try. Here, we aim to give a brief overview of the main approaches to live cell imaging, with some mention of their pros and cons.

Cell Physiological Phenomena↗
PubMed2003

A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment.

Glycogen synthase kinase-3 (GSK-3) is a conserved, multifunctional kinase that is constitutively active in resting cells, and inactivated through phosphorylation by protein kinase B (PKB). We have investigated the temporal and spatial control of GSK-3 phosphorylation during the cell cycle in mammalian cells. We show that GSK-3 is present along the length of spindle microtubules and that a fraction of GSK-3 is phosphorylated during mitosis. Phospho-GSK-3 is abundant at the centrosomes and spindle poles but absent from other areas of the spindle. GSK-3 phosphorylation occurs concomitantly with the appearance of phosphorylated and active PKB at the centrosome, which suggests that PKB is the kinase responsible for phosphorylating and inactivating GSK-3 at the centrosome during mitosis. We demonstrate that lithium and two structurally distinct inhibitors of GSK-3 promote defects in microtubule length and chromosomal alignment during prometaphase. Treated cells contain mono-oriented chromosomes concentrated at the plus ends of astral microtubules, which are longer than in untreated cells. Live microscopy of cells expressing Histone-2B-GFP confirms that the inhibition of GSK-3 suppresses mitotic chromosome movement and leads to a prometaphase-like arrest. We propose that GSK-3 is regulated in a temporal and spatial manner during mitosis and, through controlling microtubule dynamics, plays an important role in chromosomal alignment on the metaphase plate.

Cell Cycle Proteins↗