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Byline: George Patterson, Biophotonics Section, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892; Michael Davidson, National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, Florida 32310; Suliana Manley, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland CH-1015; Jennifer Lippincott-Schwartz, Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; email: lippincj@mail.nih.gov Keywords: superresolution microscopy, single molecule, PALM, STORM, FPALM, diffraction limit, photoactivation, photoactivatable fluorescent protein, fluorescence imaging Abstract Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude ([Sm]10-20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule-based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales.

The dorsoventral (DV) patterning of the Drosophila embryo depends on the nuclear localization gradient of Dorsal (DI), a protein related to the mammalian NF-[kappa]B transcription factors. Current understanding of how the DI gradient works has been derived from studies of its transcriptional interpretation, but the gradient itself has not been quantified. In particular, it is not known whether the DI gradient is stable or dynamic during the DV patterning of the embryo. To address this question, we developed a mathematical model of the DI gradient and constrained its parameters by experimental data. Based on our computational analysis, we predict that the DI gradient is dynamic and, to a first approximation, can be described as a concentration profile with increasing amplitude and constant shape. These time-dependent properties of the DI gradient are different from those of the Bicoid and MAPK phosphorylation gradients, which pattern the anterior and terminal regions of the embryo. Specifically, the gradient of the nuclear levels of Bicoid is stable, whereas the pattern of MAPK phosphorylation changes in both shape and amplitude. We attribute these striking differences in the dynamics of maternal morphogen gradients to the differences in the initial conditions and chemistries of the anterior, DV, and terminal systems. computational modeling | Drosophila | systems biology | parameter estimation doi/10.1073/pnas.0912395106

The mechanisms controlling cell shape changes within epithelial monolayers for tissue formation and reorganization remain unclear. Here, we investigate the role of dynamin, a large GTPase, in epithelial morphogenesis. Depletion of dynamin 2 (Dyn2), the only dynamin in epithelial cells, prevents establishment and maintenance of epithelial polarity, with no junctional formation and abnormal actin organization. Expression of Dyn2 mutants shifted to a non-GTP state, by contrast, causes dramatic apical constriction without disrupting polarity. This is due to Dyn2's interactions with deacetylated cortactin and downstream effectors, which cause enhanced actomyosin contraction. Neither inhibitors of endocytosis nor GTP-shifted Dyn2 mutants induce apical constriction. This suggests that GTPase-dependent changes in Dyn2 lead to interactions with different effectors that may differentially modulate endocytosis and/or actomyosin dynamics in polarized cells. We propose this enables Dyn2 to coordinate, in a GTPase-dependent manner, membrane recycling and actomyosin contractility during epithelial morphogenesis. deacetylated corcactin | actin | polarity | morphogenesis | epithelial cells doi/10.1073/pnas.0909812106

Mitochondria undergo fission-fusion events that render these organelles highly dynamic in cells. We report a relationship between mitochondrial form and cell cycle control at the [G.sub.1]-S boundary. Mitochondria convert from isolated, fragmented elements into a hyperfused, giant network at [G.sub.1]-S transition. The network is electrically continuous and has greater ATP output than mitochondria at any other cell cycle stage. Depolarizing mitochondria at early [G.sub.1] to prevent these changes causes cell cycle progression into S phase to be blocked. Inducing mitochondrial hyperfusion by acute inhibition of dynamin-related protein-1 (DRP1) causes quiescent cells maintained without growth factors to begin replicating their DNA and coincides with buildup of cyclin E, the cyclin responsible for [G.sub.1]-to-S phase progression. Prolonged or untimely formation of hyperfused mitochondria, through chronic inhibition of DRP1, causes defects in mitotic chromosome alignment and S-phase entry characteristic of cyclin E overexpression. These findings suggest a hyperfused mitochondrial system with specialized properties at [G.sub.1]-S is linked to cyclin E buildup for regulation of [G.sub.1]-to-S progression. cell cycle | mitochondrial morphology | dynamin-related protein 1

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals., The ESCRT machinery is essential for several fundamental cellular pathways (1-7). The biogenesis of lysosomes involves the maturation of early endosomes into multivesicular bodies (MVBs) (1,2,5). In this pathway, portions [...]

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy, iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture. fluorescence microscopy | interferometry | PALM | photoactivated localization microscopy | single molecule imaging

Autophagy is responsible for nonspecific, bulk degradation of cytoplasmic components. Recent work has revealed also that there is specific, autophagic degradation of polyubiquitinated protein aggregates, whose buildup occurs during neurodegenerative disease. Here, we report that simple mono-ubiquitination of normally long-lived cytoplasmic substrates is sufficient to target these substrates for autophagic degradation in mammalian cells. That is, upon their ubiquitination, both small [i.e., red fluorescent protein (RFP)] and large (i.e., peroxisomes) substrates are efficiently targeted to autophagosomes and then degraded within lysosomes upon autophagosome-lysosome fusion. This targeting requires the ubiquitin-binding protein, p62, and is blocked by the Class III phosphatidylinositol 3-kinase (PI3K) inhibitor, 3-methyladenine (3-MA), or by depletion of the autophagy-related-12 (Atg12) protein homolog. Mammalian cells thus use a common pathway involving ubiquitin and p62 for targeting diverse types of substrates for autophagy. autophagy | p62 | pexophagy

Author(s): Alex M. Valm [1]; Sarah Cohen [1]; Wesley R. Legant [2]; Justin Melunis [3]; Uri Hershberg [3, 4]; Eric Wait [5]; Andrew R. Cohen [5]; Michael W. Davidson [6]; [...]

Peroxisomes are ubiquitous organelles that proliferate under different physiological conditions and can form de novo in cells that lack them. The endoplasmic reticulum (ER) has been shown to be the source of peroxisomes in yeast and plant cells. It remains unclear, however, whether the ER has a similar role in mammalian cells and whether peroxisome division or outgrowth from the ER maintains peroxisomes in growing cells. We use a new in cellula pulse-chase imaging protocol with photo-activatable GFP to investigate the mechanism underlying the biogenesis of mammalian peroxisomes. We provide direct evidence that peroxisomes can arise de novo from the ER in both normal and peroxisome-less mutant cells. We further show that PEX16 regulates this process by being cotranslationally inserted into the ER and serving to recruit other peroxisomal membrane proteins to membranes. Finally, we demonstrate that the increase in peroxisome number in growing wild-type cells results primarily from new peroxisomes derived from the ER rather than by division of preexisting peroxisomes.

The folding environment in the endoplasmic reticulum (ER) depends on multiple abundant chaperones that function together to accommodate a range of substrates. The ways in which substrate engagement shapes either specific chaperone dynamics or general ER attributes in vivo remain unknown. In this study, we have evaluated how changes in substrate flux through the ER influence the diffusion of both the lectin chaperone calreticulin and an inert reporter of ER crowdedness. During acute changes in substrate load, the inert probe revealed no changes in ER organization, despite significant changes in calreticulin dynamics. By contrast, inhibition of the lectin chaperone system caused rapid changes in the ER environment that could be reversed over time by easing new substrate burden. Our findings provide insight into the normal organization and dynamics of an ER chaperone and characterize the capacity of the ER to maintain homeostasis during acute changes in chaperone activity and availability. castanospermine | fluorescence loss in photobleaching | fluorescence recovery after photobleaching | pactamycin | puromycin

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.

Hepatocytes polarize by forming functionally distinct sinusoidal (basolateral) and canalicular (apical) plasma membrane domains. Two distinct routes are used for delivery of membrane proteins to the canaliculus. Proteins having glycosylphosphatidylinositol anchors or single transmembrane domains are targeted to the sinusoidal plasma membrane from where they transcytose to the canalicular domain. In contrast, apical ATP-binding-cassette (ABC) transporters, which are required for energy-dependent biliary secretion of bile acids (ABCB11), phospholipids (ABCB4), and nonbile acid organic anions (ABCC2), lack initial residence in the basolateral plasma membrane and traffic directly from Golgi membranes to the canalicular membrane. While investigating mechanisms of apical targeting in WIF-B9 cells, a polarized hepatic epithelial cell line, we observed that rab11a is required for canalicular formation. Knockdown of rab11a or overexpression of the rab11a-GDP locked form prevented canalicular formation as did overexpression of the myosin Vb motorless tail domain. In WIF-B9 cells, which lack bile canaliculi, apical ABC transporters colocalized with transcytotic membrane proteins in rab11a-containing endosomes and, unlike the transcytotic markers, did not distribute to the plasma membrane. We propose that polarization of hepatocytes (i.e., canalicular biogenesis) requires recruitment of rab11a and myosin Vb to intracellular membranes that contain apical ABC transporters and transcytoticf markers, permitting their targeting to the plasma membrane. In this model, polarization is initiated upon delivery of rab11a-myosin Vb-containing membranes to the surface, which causes plasma membrane at the site of delivery to differentiate into apical domain (bile canaliculus). hepatocyte polarization | plasma membrane segregation

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/ Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with ~40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 [micro]m), exhibiting Ds that varied 1 0-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

Protein translocons of the mammalian endoplasmic reticulum are composed of numerous functional components whose organization during different stages of the transport cycle in vivo remains poorly understood. We have developed generally applicable methods based on fluorescence resonance energy transfer (FRET) to probe the relative proximities of endogenously expressed translocon components in cells. Examination of substrate-engaged translocons revealed oligomeric assemblies of the Sec61 complex that were associated to varying degrees with other essential components including the signal recognition particle receptor TRAM and the TRAP complex. Remarkably, these components not only remained assembled but also had a similar, yet distinguishable, organization both during and after nascent chain translocation. The persistence of preassembled and complete translocons between successive rounds of transport may facilitate highly efficient translocation in vivo despite temporal constraints imposed by ongoing translation and a crowded cellular environment.

In mitosis, chromosome, cytoskeleton, and organelle dynamics must be coordinated for successful cell division. Here, we present evidence for a role for Arf1, a small GTPase associated with the Golgi apparatus, in the orchestration of mitotic Golgi breakdown, chromosome segregation, and cytokinesis. We show that early in mitosis Arf1 becomes inactive and dissociates from Golgi membranes. This is followed by the dispersal of numerous Arf1-dependent peripheral Golgi proteins and subsequent Golgi disassembly. If Arf1 is kept in an active state by treatment with the small molecule H89 or expression of its GTP-locked form, intact Golgi membranes with bound peripheral proteins persist throughout mitosis. These cells enter mitosis but exhibit gross defects in chromosome segregation and cytokinetic furrow ingression. These findings suggest that mitotic Golgi disassembly depends on Arf1 inactivation and is used by the cell to disperse numerous peripheral Golgi proteins for coordinating the behavior of Golgi membranes, chromosomes, and cytoskeleton during mitosis.

The endoplasmic reticulum (ER) can transform from a network of branching tubules into stacked membrane arrays (termed organized smooth ER [OSER]) in response to elevated levels of specific resident proteins, such as cytochrome b(5). Here, we have tagged OSER-inducing proteins with green fluorescent protein (GFP) to study OSER biogenesis and dynamics in living cells. Overexpression of these proteins induced formation of karmellae, whorls, and crystalloid OSER structures. Photobleaching experiments revealed that OSER-inducing proteins were highly mobile within OSER structures and could exchange between OSER structures and surrounding reticular ER. This indicated that binding interactions between proteins on apposing stacked membranes of OSER structures were not of high affinity. Addition of GFP, which undergoes low affinity, antiparallel dimerization, to the cytoplasmic domains of non-OSER-inducing resident ER proteins was sufficient to induce OSER structures when overexpressed, but addition of a nondimerizing GFP variant was not. These results point to a molecular mechanism for OSER biogenesis that involves weak homotypic interactions between cytoplasmic domains of proteins. This mechanism may underlie the formation of other stacked membrane structures within cells.

The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells., The development of fluorescent proteins as molecular tags over the past decade has spurred a revolution by allowing complex biochemical processes to be correlated with the functioning of proteins in [...]

We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for clays under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange., Photoactivation, the rapid conversion of photoactivatable molecules to a fluorescent state by intense irradiation, can be used to mark and monitor selected molecules within cells (1). Previous efforts to develop [...]

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyl-transferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP-Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP-Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant ([D.sub.eff]) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP-Dad1 in the ER membranes, but to a level that is still lower than that of free GFP-Dad1. This suggests that GFP--Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.

Author(s): John F. Presley [1, 2]; Theresa H. Ward [1]; Andrea C. Pfeifer [1]; Eric D. Siggia [3]; Robert D. Phair [4]; Jennifer Lippincott-Schwartz (corresponding author) [1] Cytosolic coat proteins [...]

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1 [T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1 [H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1 [T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1--COPII and Arf1--coatomer systems.

Research has been conducted on the Golgi complex functions. The regulation of the cargo protein inclusion and vesicle budding by the Golgi complex has been investigated.

Results show that during mitotic cell division Golgi membranes disperse and reform across endoplasm reticulum, maximally using constitutive recycling pathways. Data further indicate that endoplasm reticulum plays an essential role in the Golgi coplex's genesis and inheritance.

Endoplasmic reticulum (ER)-specific chaperones and folding enzymes facilitate folding and unfolding reactions of the newly synthesized proteins in the ER and are known to serve a quality control function, allowing only correctly assembled and folded proteins to exit the compartment. A new study investigates the extent to which proteins leaving the ER return to its folding environment from the Golgi complex. It is concluded that recycling is not induced by misfolded proteins and is not signal-mediated, but could be an inherent property of proteins within the Golgi membranes.

Nuclear membrane dynamics and assembly in living cells during interphase and mitosis were analyzed using the inner nuclear membrane protein, lamin B receptor (LBR), fused with green fluorescent protein (GFP). Results show a rapid and free distribution of LBR-GFP in ER membranes of interphase cells. Moreover, LBR-GFP localization in the inner nuclear membrane reveals complete immobilization which demonstrate its tight binding in heterochromatin and/or the lamina. Findings in mitotic cells, however, contradict the vesiculation model for the disassembly and reassembly of NE membrane. This suggests that processes during interphase can also illustrate mitotic localization of inner nuclear proteins.

Proteins from the endoplasmic reticulum (ER) are channelled through the Golgi complex before being transported to their final destinations. It is now possible to follow the entire life history of a single transport intermediates in living cells, through new florescent tagging techniques. ER-to-Golgi transport has been visualized through the use of tso45 VSVG, a viral glycoprotein, tagged with green fluorescent protein.

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.sup.-9] square centimeters per second to 5 x [10.sup.-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., The Golgi complex contains a large number of resident components that play important roles in the processing and sorting of secretory and membrane proteins, but how these components are maintained [...]

Various microscopic and microinjection techniques help study the distribution and function of the microtubule motor kinesin in membrane structures comprising the ER/Golgi system. Microscopic techniques locate kinesin on all membranes of the ER/Glogi system, peripherally directing the movements of the membrane in that region. Microinjection studies with kinesin antibody indicate kinesin to be involved in transport from Glogi-to-ER but not from Er-to-Glogi.

The most striking effect of brefeldin A is its influence on cellular membrane transport. It alters the form and origin of some components of the vacuolar system of cells. This is attributed to the drug's inhibitory effect on the binding of coat proteins to their target organelles. The regulation of membrane traffic through specific coat proteins and the structural modifications responsible for the transport of materials across the cell membrane are also reviewed.

The article discusses the development of a new bright monomeric photoactivabale red fluorescent protein, which can be employed for the two-color super-resolution of single particle tracking PALM of live cells. The protein is shown to exhibit better brightness and stability, as compared to the other monomeric proteins.

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 β-COP. Addition of βγ subunits of G proteins inhibited the association of both ARF and β-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-y-S). Thus, the heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane., CYTOSOLIC PROTEINS THAT BIND REversibly to membranes of the Golgi complex have been identified [1, 2] and shown to exist as a high molecular weight complex in the cytosol referred [...]

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle, Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153)in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus. GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 [degrees] C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells. Key words: Golgi complex * endocytes * glycosyl phosphatidylinositol anchor * raft * green fluorescent protein

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sari-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites. Key words: Sar1 * COPII * endoplasmic reticulum * vesicle * cargo export

Author(s): Jennifer Lippincott-Schwartz (corresponding author); Wei Liu Many cells use small, membrane-bounded carriers -- vesicles -- to release and take up molecules and to move proteins between membrane-clad intracellular compartments. [...]

Author(s): Jennifer Lippincott-Schwartz (corresponding author) One of the many events that take place as cells divide is the breakdown of the nuclear envelope -- the membrane that surrounds the nucleus. [...]

Quantitative time-lapsed imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG-GFP), were utilized for kinetic modeling of protein traffic through the different compartments of the secretory pathway. A series of first order rate laws was able to accurately represent VSVG-GFP transport and allowed the calculation of compartment residence times and rate constants for movement into and out of the Golgi complex and delivery to the plasma membrane.

Fluorescence imaging illuminated on the link between the nonreceptor protein tyrosine kinase ZAP-70 and T cell receptor (TCR) zeta. Analysis revealed that the stimulation-dependent translocation of ZAP-70 to the cell surface in HeLa cells relied on expression of a chimeric TCR zeta chain. This ZAP-70 translocation was improved through the co-expression of active Lck. In addition, relocated ZAP-70 was found to be specifically linked to the chimeric zeta chain. Photobleaching showed that cytosolic and nuclear ZAP-70 is highly mobile and freely diffusible, and that it transforms into a more static state when it translocates to the cell periphery.