Transport Through the Secretory Pathway: Observations of Cargo and Peripheral Coat Proteins

We have used green fluorescent protein (GFP) chimeras to examine the dynamics of the early secretory pathway and the role of the peripheral coat protein COP I. To describe the overall properties of ER to Golgi transport we used the temperature sensitive viral glycoprotein, ts045 VSVG, tagged with GFP at its cytoplasmic tail. VSVG-GFP retained the temperature sensitive phenotype of its parent: it reversibly misfolded and was retained in the ER at 40°C. Upon shift to 32°C it was rapidly exported from the ER, moving as a synchronous pool into the Golgi complex and then to the cell surface. Using time-lapse imaging of living cells expressing VSVG-GFP we found that the carriers for ER to Golgi traffic are tubulovesicular pre-Golgi intermediates that move centrosomally to the Golgi at speeds of>1 μM2/sec and then fuse with the cis face of the Golgi complex. These movements are dependant on microtubules and the dynein/dynactin complex.

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Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

Journal of Cell Science ◽

10.1242/jcs.113.18.3151 ◽

2000 ◽

Vol 113 (18) ◽

pp. 3151-3159 ◽

Cited By ~ 6

Author(s):

R. Blum ◽

D.J. Stephens ◽

I. Schulz

Keyword(s):

Fluorescent Protein ◽

Time Lapse ◽

Temperature Sensitive ◽

Long Distance ◽

Anterograde Transport ◽

Network Transport ◽

Vesicular Stomatitis ◽

Green Fluorescent ◽

Time Lapse Imaging ◽

Tubular Membranes

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www.biologists.com/JCS/movies/jcs1334.html

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Retrograde Transport of Golgi-localized Proteins to the ER

The Journal of Cell Biology ◽

10.1083/jcb.140.1.1 ◽

1998 ◽

Vol 140 (1) ◽

pp. 1-15 ◽

Cited By ~ 153

Author(s):

Nelson B. Cole ◽

Jan Ellenberg ◽

Jia Song ◽

Diane DiEuliis ◽

Jennifer Lippincott-Schwartz

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Fusion Proteins ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Viral Glycoprotein ◽

Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

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Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules

Microscopy and Microanalysis ◽

10.1017/s1431927600007583 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 139-140

Author(s):

John Presley ◽

Koret Hirschberg ◽

Nelson Cole

Keyword(s):

Green Fluorescent Protein ◽

Membrane Transport ◽

G Protein ◽

Golgi Complex ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Dynamic Properties ◽

Living Cells ◽

Morphological Properties ◽

Green Fluorescent

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

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Golgi-bound Rab34 Is a Novel Member of the Secretory Pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e06-11-0991 ◽

2007 ◽

Vol 18 (12) ◽

pp. 4762-4771 ◽

Cited By ~ 29

Author(s):

Neil M. Goldenberg ◽

Sergio Grinstein ◽

Mel Silverman

Keyword(s):

Hela Cells ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Brefeldin A ◽

Fluid Phase ◽

Dominant Negative ◽

Temperature Sensitive ◽

Golgi Stack ◽

Green Fluorescent ◽

Intracellular Vesicle Transport

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

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The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

Molecular Biology of the Cell ◽

10.1091/mbc.9.7.1741 ◽

1998 ◽

Vol 9 (7) ◽

pp. 1741-1756 ◽

Cited By ~ 88

Author(s):

Jason A. Kahana ◽

Gabriel Schlenstedt ◽

Darren M. Evanchuk ◽

John R. Geiser ◽

M. Andrew Hoyt ◽

...

Keyword(s):

Mitotic Spindle ◽

Fluorescent Protein ◽

Time Lapse ◽

Cytoplasmic Dynein ◽

Stable Complex ◽

Complex Protein ◽

Mother And Daughter ◽

Green Fluorescent ◽

Dynactin Complex

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene,NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150 glued . Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

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Factor VIII-eGFP fusion proteins with preserved functional activity for the analysis of the early secretory pathway of factor VIII

Thrombosis and Haemostasis ◽

10.1160/th08-12-0807 ◽

2009 ◽

Vol 102 (11) ◽

pp. 925-935 ◽

Cited By ~ 3

Author(s):

Stefan Heinz ◽

Jörg Schüttrumpf ◽

Jeremy Simpson ◽

Rainer Pepperkok ◽

Gerry Nicolaes ◽

...

Keyword(s):

Factor Viii ◽

Fusion Proteins ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Recombinant Expression ◽

Early Secretory Pathway ◽

C Terminus ◽

Living Organisms ◽

Green Fluorescent

SummaryConsidering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.

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Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

Applied and Environmental Microbiology ◽

10.1128/aem.06644-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8310-8317 ◽

Cited By ~ 27

Author(s):

Joshua D. Morris ◽

Jessica L. Hewitt ◽

Lawrence G. Wolfe ◽

Nachiket G. Kamatkar ◽

Sarah M. Chapman ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescent Protein ◽

Nile Red ◽

Temporal Distribution ◽

Time Lapse ◽

Content Type ◽

Rhamnolipid Production ◽

Serovar Typhimurium ◽

Surface Motility ◽

Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

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Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽

10.1182/blood-2010-01-264382 ◽

2010 ◽

Vol 116 (6) ◽

pp. 909-914 ◽

Cited By ~ 114

Author(s):

Enid Yi Ni Lam ◽

Christopher J. Hall ◽

Philip S. Crosier ◽

Kathryn E. Crosier ◽

Maria Vega Flores

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Fluorescent Protein ◽

Living Organism ◽

Time Lapse ◽

Dorsal Aorta ◽

Transgenic Zebrafish ◽

Hematopoietic Stem ◽

Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0104 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4622-4632 ◽

Cited By ~ 54

Author(s):

Yasmina Bauer ◽

Philipp Knechtle ◽

Jürgen Wendland ◽

Hanspeter Helfer ◽

Peter Philippsen

Keyword(s):

Fluorescent Protein ◽

Hyphal Growth ◽

Time Lapse ◽

Growth Direction ◽

Ashbya Gossypii ◽

Growth Guidance ◽

Characteristic Features ◽

Green Fluorescent ◽

Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

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Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes

Blood ◽

10.1182/blood-2005-06-2204 ◽

2005 ◽

Vol 106 (13) ◽

pp. 4076-4085 ◽

Cited By ~ 142

Author(s):

Sunita R. Patel ◽

Jennifer L. Richardson ◽

Harald Schulze ◽

Eden Kahle ◽

Niels Galjart ◽

...

Keyword(s):

Fluorescent Protein ◽

Average Rate ◽

Blood Platelets ◽

Time Lapse ◽

Microtubule Assembly ◽

Alternative Mechanism ◽

Differentiated Cells ◽

Continuous Polymerization ◽

Regulatory Complex ◽

Green Fluorescent

Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 μm/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plus-end marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3–green fluorescent protein (GFP)–expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 μm/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 μm/min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100–permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 μm/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactindynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a vital component of proplatelet elongation.

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