Golgi Marker Research Articles

Identification and characterisation of a cryptic Golgi complex in Naegleria gruberi.

ABSTRACTAlthough the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in Naegleria, one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of Naegleria and show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker (Ng)CopB by fluorescence in Naegleria gruberi, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that NgCOPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.

Epstein-Barr Virus Acquires Its Final Envelope on Intracellular Compartments With Golgi Markers.

Herpesvirus subfamilies typically acquire their final envelope in various cytoplasmic compartments such as the trans-Golgi network (TGN), and endosomes prior to their secretion into the extracellular space. However, the sites for the final envelopment of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, are poorly understood. Here, we characterized the sites for the final envelopment of EBV in Burkitt’s lymphoma cell lines induced into the lytic cycle by crosslinking cell surface IgG. Electron microscopy revealed the various stages of maturation and egress of progeny virions including mature EBV in irregular cytoplasmic vesicles. Immunofluorescence staining showed that gp350/220, the major EBV glycoprotein, and the viral capsid antigen, p18, efficiently colocalized with a cis-Golgi marker, GM130. gp350/220 partly colocalized with the TGN, which was distributed in a fragmented and dispersed pattern in the cells induced into the lytic cycle. In contrast, limited colocalization was observed between gp350/220 and endosomal markers, such as a multi-vesicular bodies marker, CD63, a recycling endosome marker, Rab11, and a regulatory secretion vesicles marker, Rab27a. Finally, we observed that treatment of cells with brefeldin A, an inhibitor of vesicle trafficking between the endoplasmic reticulum and Golgi apparatus, resulted in the perinuclear accumulation of gp350/220 and inhibition of its distribution to the plasma membrane. Brefeldin A also inhibited the release of infectious EBV. Taken together, our findings support a model in which EBV acquires its final envelope in intracellular compartments containing markers of Golgi apparatus, providing new insights into how EBV matures.

623: Decreased myometrial expression of matrix-Gla protein (MGP) is associated with preterm and term laboring state

Uterine contractions are driven by complex calcium mediated mechanisms. Matrix Gla-protein (MGP) is an emerging vitamin K2-dependent protein with key roles in calcium homeostasis and inhibition of smooth muscle calcification. We sought to study MGP expression to provide new insight into the physiology of term and preterm contracting human myometrium. Myometrial expression of MGP was investigated in the following groups: 1) term labor (TL, n=5, GA: 39); 2) term non-labor (TNL, n=5, GA: 39); 3) preterm labor (PTL, n=5, GA: 28); 4) preterm non-labor (PTNL, n=13, GA: 29). Serum levels of MGP were measured by ELISA. Myometrial expression was assessed using immunohistochemistry and RNAseq with qRT-PCR validation. MGP and calcium-related proteins were localized with immunofluorescence in pregnant (PHM1) and non-pregnant (hTERT) human myometrial cell lines. 1) Pregnant myometrial cell lines had greater expression of MGP compared to non-pregnant state (Figure A). 2) At term, maternal serum MGP levels were unaffected by labor. 3) Cytoplasm of human myocytes display MGP immunostaining constitutively. 4) By qRT-PCR, we identified significantly higher levels of MGP gene expression in non-laboring myometrium compared to laboring preterm (p=0.002) and term (p=0.0004) tissues (Figure B). 5) This expression pattern was paralleled in mRNAseq of term (p=0.003) and preterm (p=0.002) uterus. 6) Immunocytochemistry showed that MGP colocalized with smooth-muscle actin monofilaments, but not calcium exchangers (SERCA, PMCA1) and Golgi marker. Our study presents the novel findings that MGP is present in human myometrium, and its tissue withdrawal is associated with a laboring state.

The plant membrane surrounding powdery mildew haustoria shares properties with the endoplasmic reticulum membrane.

Many filamentous plant pathogens place specialized feeding structures, called haustoria, inside living host cells. As haustoria grow, they are believed to manipulate plant cells to generate a specialized, still enigmatic extrahaustorial membrane (EHM) around them. Here, we focused on revealing properties of the EHM. With the help of membrane-specific dyes and transient expression of membrane-associated proteins fused to fluorescent tags, we studied the nature of the EHM generated by barley leaf epidermal cells around powdery mildew haustoria. Observations suggesting that endoplasmic reticulum (ER) membrane-specific dyes labelled the EHM led us to find that Sar1 and RabD2a GTPases bind this membrane. These proteins are usually associated with the ER and the ER/cis-Golgi membrane, respectively. In contrast, transmembrane and luminal ER and Golgi markers failed to label the EHM, suggesting that it is not a continuum of the ER. Furthermore, GDP-locked Sar1 and a nucleotide-free RabD2a, which block ER to Golgi exit, did not hamper haustorium formation. These results indicated that the EHM shares features with the plant ER membrane, but that the EHM membrane is not dependent on conventional secretion. This raises the prospect that an unconventional secretory pathway from the ER may provide this membrane's material. Understanding these processes will assist future approaches to providing resistance by preventing EHM generation.

Cell type-dependent localization of MLO proteins

ABSTRACTMildew resistance locus O (MLO) proteins are transmembrane proteins that mediate cell-cell communication in plants. We recently demonstrated the importance of subcellular localization to MLO function during pollen tube reception. NORTIA (NTA), the MLO protein involved in this process, localizes to the Golgi of the synergid cell before interaction with the pollen tube. MLO proteins that can substitute for NTA's function in this pathway all partially localize with the same Golgi marker in the synergid cell. In this study, we report that MLO subcellular localization is cell type-dependent, with different distributions of some MLOs observed when ectopically expressed in the epidermal cells of tobacco and Arabidopsis compared to synergids. This dependency may be due to co-factors that influence MLO function within a given cell type, providing an intriguing new target for understanding MLO distribution and subsequent function in their respective processes.

Knockout of the gamma subunit of the AP-1 adaptor complex in the human parasite Trypanosoma cruzi impairs infectivity and differentiation and prevents the maturation and targeting of the major protease cruzipain.

The AP-1 Adaptor Complex assists clathrin-coated vesicle assembly in the trans-Golgi network (TGN) of eukaryotic cells. However, the role of AP-1 in the protozoan Trypanosoma cruzi—the Chagas disease parasite—has not been addressed. Here, we studied the function and localization of AP-1 in different T. cruzi life cycle forms, by generating a gene knockout of the large AP-1 subunit gamma adaptin (TcAP1-γ), and raising a monoclonal antibody against TcAP1-γ. Co-localization with a Golgi marker and with the clathrin light chain showed that TcAP1-γ is located in the Golgi, and it may interact with clathrin in vivo, at the TGN. Epimastigote (insect form) parasites lacking TcAP1-γ (TcγKO) have reduced proliferation and differentiation into infective metacyclic trypomastigotes (compared with wild-type parasites). TcγKO parasites have also displayed significantly reduced infectivity towards mammalian cells. Importantly, TcAP1-γ knockout impaired maturation and transport to lysosome-related organelles (reservosomes) of a key cargo—the major cysteine protease cruzipain, which is important for parasite nutrition, differentiation and infection. In conclusion, the defective processing and transport of cruzipain upon AP-1 ablation may underlie the phenotype of TcγKO parasites.

Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering.

The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.

Biological responses of osteocytic connexin 43 hemichannels to simulated microgravity.

Connexin 43 (Cx43) hemichannels and gap junctions in osteocytes are responsive to mechanical loading, which is important for bone formation and remodeling. However, the mechanism of these Cx43-forming channels in the process of mechanical unloading is still not very clear. In this study, unloading caused by weightlessness was simulated by using a random position machine (RPM). Osteocytic MLO-Y4 cells were subjected to 2 h of RPM treatment, and levels of Cx43 mRNA and total and cell surface expressed protein were determined by quantitative real-time PCR, western blotting, and biotinylation analysis. Although mRNA was elevated by RPM, total protein level of Cx43 was not altered; however, surface biotinylated Cx43 was significantly reduced. Interestingly, RPM promoted the retention of Cx43 in the Golgi apparatus detected by co-immunofluorescence with antibodies against Cx43 and 58 K Golgi marker protein. Dye uptake assay showed that hemichannels were induced open after RPM for 2 h. Consistently, prostaglandin E2 release was increased and this increase was completely attenuated with the treatment of a Cx43 hemichannel blocking antibody. Together, this study demonstrates increased activity of Cx43 hemichannels to RPM, and active Cx43 hemichannels with prostaglandin E2 release are likely to module biological function under simulated weightless conditions. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1195-1202, 2017.

Subcellular Localization of Arabidopsis Pathogenesis-Related 1 (PR1) Protein.

The Arabidopsis thaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.

Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3− cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3− cells displayed delayed streaming and aggregation, and interestingly, cln3− cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3− CM, of which 3 were absent in cln3− CM. Moreover, 12 proteins that were present in cln3− CM were absent in WT CM. Label-free quantification identified 42 proteins that were present in significantly higher amounts in cln3− CM compared to WT, and 3 proteins that were present in significantly reduced amounts. A GO term enrichment analysis showed that a majority of the affected proteins are linked to endocytosis, vesicle-mediated transport, proteolysis, and metabolism. In total, the results of this study indicate that Cln3 functions in both conventional and unconventional protein secretion and that loss of Cln3 results in deregulated secretion during early development. Importantly, this is the first evidence in any system linking CLN3 function to protein secretion.

Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.

Fixation methods can differentially affect ciliary protein immunolabeling

BackgroundPrimary cilia are immotile, microtubule-based organelles present on most cells. Defects in primary cilia presence/function result in a category of developmental diseases referred to as ciliopathies. As the cilia field progresses, there is a need to consider both the ciliary and extraciliary roles of cilia proteins. However, traditional fixation methods are not always suitable for examining the full range of localizations of cilia proteins. Here, we tested a variety of fixation methods with commonly used cilia markers to determine the most appropriate fixation method for different cilia proteins.MethodsMouse inner medullary collecting duct and human retinal pigmented epithelial cells were grown to confluence, serum starved, and fixed with one of the following fixation agents: paraformaldehyde–sucrose, paraformaldehyde–PBS, methanol, cytoskeletal buffer followed by methanol, or three variations of cytoskeletal buffer–paraformaldehyde fixation. Each cell type and fixation method combination was probed with the following ciliary markers: acetylated α-tubulin, detyrosinated tubulin, polyglutamylated tubulin, β-tubulin, adenylyl cyclase 3 (AC3), ADP-ribosylation factor-like protein 13b (Arl13b), centrosome and spindle pole associated protein 1 (CSPP1), or intraflagellar transport protein 20 (IFT20). Intraflagellar transport protein 88 (IFT88) and GM130 (Golgi marker) were also used. We assessed actin (via phalloidin) and microtubule integrity, centrioles, cilia, and two extraciliary sites (mitotic figures and Golgi).ResultsFor the cilia markers examined, paraformaldehyde fixation preserved cilia immunolabeling of cilia-membrane proteins (AC3 and Arl13b), but failed to reveal cilia immunostaining of axonemal proteins (CSPP1 and IFT20). Methanol revealed cilia labeling for some axonemal proteins, but not others, and this depended on cell type. Generally, any method that first included a wash in cytoskeletal buffer, before fixing, revealed more distinct cilia immunolabeling for axonemal proteins (CSPP1, IFT20, and IFT88), but resulted in the loss of cilia labeling for cilia-membrane proteins (AC3 and Arl13b). All three different post-translational modifications of tubulin antibodies positively immunolabeled cilia in all fixation methods tested. Ultimately, we found that fixing cells in a solution of paraformaldehyde prepared in cytoskeletal buffer allowed for the preservation of cilia immunolabeling for most cilia proteins tested and allowed visualization of two extraciliary sites (mitotic figures and Golgi).ConclusionSome general patterns were observed to guide in the choice of a fixation agent. Cilia-membrane proteins generally benefit from quick fixation with no prior permeabilization, whereas axonemal proteins tend to benefit from permeabilization and use of cytoskeletal buffer.

The trans-Golgi Network and the Golgi Stacks Behave Independently During Regeneration After Brefeldin A Treatment in Tobacco BY-2 Cells.

The trans-Golgi network (TGN) plays an essential role in intracellular membrane trafficking. In plant cells, recent live-cell imaging studies have revealed the dynamic behavior of the TGN independent from the Golgi apparatus. In order to better understand the relationships between the two organelles, we examined their dynamic responses to the reagent brefeldin A (BFA) and their recovery after BFA removal. Golgi markers responded to BFA similarly over a range of concentrations, whereas the behavior of the TGN was BFA concentration dependent. The TGN formed aggregates at high concentrations of BFA; however, TGN proteins relocalized to numerous small vesicular structures dispersed throughout the cytoplasm at lower BFA concentrations. During recovery from weak BFA treatment, the TGN started to regenerate earlier than the completion of the Golgi. The regeneration of the two organelles proceeded independently of each other for a while, and eventually was completed by their association. Our data suggest that there is some degree of autonomy for the regeneration of the TGN and the Golgi in tobacco BY-2 cells.

SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome.

A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activity of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signalling.

Putative Membrane-Bound Transporters MFSD14A and MFSD14B Are Neuronal and Affected by Nutrient Availability.

Characterization of orphan transporters is of importance due to their involvement in cellular homeostasis but also in pharmacokinetics and pharmacodynamics. The tissue and cellular localization, as well as function, is still unknown for many of the solute carriers belonging to the major facilitator superfamily (MFS) Pfam clan. Here, we have characterized two putative novel transporters MFSD14A (HIAT1) and MFSD14B (HIATL1) in the mouse central nervous system and found protein staining throughout the adult mouse brain. Both transporters localized to neurons and MFSD14A co-localized with the Golgi marker Giantin in primary embryonic cortex cultures, while MFSD14B staining co-localized with an endoplasmic retention marker, KDEL. Based on phylogenetic clustering analyses, we predict both to have organic substrate profiles, and possible involvement in energy homeostasis. Therefore, we monitored gene regulation changes in mouse embryonic primary cultures after amino acid starvations and found both transporters to be upregulated after 3 h of starvation. Interestingly, in mice subjected to 24 h of food starvation, both transporters were downregulated in the hypothalamus, while Mfsd14a was also downregulated in the brainstem. In addition, in mice fed a high fat diet (HFD), upregulation of both transporters was seen in the striatum. Both MFSD14A and MFSD14B were intracellular neuronal membrane-bound proteins, expressed in the Golgi and Endoplasmic reticulum, affected by both starvation and HFD to varying degree in the mouse brain.

The monomeric GTPase RabA2 is required for progression and maintenance of membrane integrity of infection threads during root nodule symbiosis.

Progression of the infection canal that conducts rhizobia to the nodule primordium requires a functional Rab GTPase located in Golgi/trans-Golgi that also participate in root hair polar growth. Common bean (Phaseolus vulgaris) symbiotically associates with its partner Rhizobium etli, resulting in the formation of root nitrogen-fixing nodules. Compatible bacteria can reach cortical cells in a tightly regulated infection process, in which the specific recognition of signal molecules is a key step to select the symbiotic partner. In this work, we show that RabA2, a monomeric GTPase from common bean, is required for the progression of the infection canal, referred to as the infection thread (IT), toward the cortical cells. Expression of miss-regulated mutant variants of RabA2 resulted in an increased number of abortive infection events, including bursting of ITs and a reduction in the number of nodules. Nodules formed in these plants were small and contained infected cells with disrupted symbiosome membranes, indicating either early senescence of these cells or defects in the formation of the symbiosome membrane during bacterial release. RabA2 localized to mobile vesicles around the IT, but mutations that affect GTP hydrolysis or GTP/GDP exchange modified this localization. Colocalization of RabA2 with ArfA1 and a Golgi marker indicates that RabA2 localizes in Golgi stacks and the trans-Golgi network. Our results suggest that RabA2 is part of the vesicle transport events required to maintain the integrity of the membrane during IT progression.

Clathrin localization and dynamics in Aspergillus nidulans.

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH-GFP) which localized to three distinct subcellular structures: late Golgi (trans-Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the "endocytic collar"); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin "hubs" dispersed in synchrony after the late Golgi marker PHOSBP . Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4-64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.

Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures

Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown.Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte.In conclusion, we suggest that these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research.

Hepatitis C Virus Is Released via a Noncanonical Secretory Route.

The goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which resulted in the production of fluorescently labeled viral particles. We used live-cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap of Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point toward the possible existence of a not-yet-described cellular secretion route.

Three-dimensional and immune electron microscopic analysis of the secretory pathway in Saccharomyces cerevisiae.

Until now, the mechanisms of ER-to-Golgi and intra-Golgi transport remain obscure. This is especially evident for the Golgi of S. cerevisiae where different Golgi compartments are not organized in stacks. Here, using improved sample preparation protocols, we examined the 3D organization of pre-Golgi and Golgi compartments and found several new features of the structures functioning along the secretory pathway. In the cytoplasmic sheet ER, we found narrow pores that aggregated near the rims, and tubular networks tightly interconnected with sheets of several cytoplasmic ER cisternae. Within the Golgi compartments, we found disks with wide pores, disks with narrow pores, and disk-like networks with varicosities or nodules at the point of branching and thick membranes. Sometimes, these compartments contained 30nm buds coated with a clathrin-like coat. The lumen of these Golgi compartments was more osmiophilic than the lumen of the ER. In contrast to ER elements, Golgi compartments were isolated and in the majority of cases not connected, although we observed some connections between Golgi compartments and also between Golgi disks with wide pores and the ER. Two types of free vesicles of 35-40 and 45-50nm were found, the former being sometimes partially coated with a clathrin-like coat. Sec31, a COPII component, was found near narrow pores in the cytoplasmic sheets of the ER, over edge aggregates of narrow pores, and within the ER network. The cis-Golgi marker Rer1p was detected on disks or semi-spheres with wide pores, while the medial Golgi marker Gos1p was found on disks or semi-spheres with narrow pores. Gos1p was found to be enriched on 45-50nm vesicles, while Rer1p was depleted. The 35-40nm vesicles did not show either label. These findings are discussed from the point of view of mechanisms of transport.