scholarly journals Visualizing Wnt secretion from Endoplasmic Reticulum to Filopodia

2018 ◽  
Author(s):  
Naushad Moti ◽  
Jia Yu ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
David M Virshup
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Signaling Cascades ◽  
Downstream Signaling ◽  
Cell Compartments ◽  
A Cell ◽  
Cortical Regions ◽  
Rate Limiting ◽  
Wnt Signal

AbstractWnts are a family of secreted palmitoleated glycoproteins that play a key role in cell to cell communications during development and regulate stem cell compartments in adults. Wnt receptors, downstream signaling cascades and target pathways have been extensively studied while less is known about how Wnts are secreted and move from producing cells to receiving cells. We used the synchronization system called Retention Using Selective Hook (RUSH) to study Wnt trafficking from endoplasmic reticulum to Golgi and then to plasma membrane and filopodia in real time. Consistent with prior studies, inhibition of porcupine (PORCN) or knockout of Wntless (WLS) blocked Wnt exit from the ER. Indeed, WLS was rate-limiting for Wnt ER exit. Wnt-containing vesicles paused at sub-cortical regions of the plasma membrane before exiting the cell. Wnt-containing vesicles were transported to adjacent cells associated with filopodia. Increasing the number of filopodia by expression of LGR5 in the producing cell increased the ability of a cell to send a Wnt signal. The RUSH system is a powerful tool to provide new insights into the Wnt secretory pathway.

scholarly journals Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

scholarly journals Life and Death of Fungal Transporters Under the Challenge of Polarity

2020 ◽  
Author(s):  
Sofia Dimou ◽  
George Diallinas
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Polarity ◽  
Secretory Pathway ◽  
Fungal Growth ◽  
Present Evidence ◽  
Life And Death ◽  
Early Endosomes ◽  
Stress Signals

Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental or stress signals. Sorting of transporters from their site of synthesis, the Endoplasmic Reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the MVB/lysosomes/vacuole system. In specific cases internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review we present evidence that shows that transporter traffic to the PM takes place through Golgi-bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking.

scholarly journals RAB6 and microtubules restrict protein secretion to focal adhesions

2019 ◽  
Vol 218 (7) ◽  
pp. 2215-2231 ◽  
Author(s):  
Lou Fourriere ◽  
Amal Kasri ◽  
Nelly Gareil ◽  
Sabine Bardin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

scholarly journals Sequestration of Mutated α1-Antitrypsin into Inclusion Bodies Is a Cell-protective Mechanism to Maintain Endoplasmic Reticulum Function

2008 ◽  
Vol 19 (2) ◽  
pp. 572-586 ◽  
Author(s):  
Susana Granell ◽  
Giovanna Baldini ◽  
Sameer Mohammad ◽  
Vanessa Nicolin ◽  
Paola Narducci ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Inclusion Bodies ◽  
Secretory Pathway ◽  
Hepatoma Cells ◽  
Neuroendocrine Cells ◽  
Protective Mechanism ◽  
High Tendency ◽  
A Cell ◽  
Cell Type Specific ◽  
Protein Disulfide

A variant α1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, α1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated α1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated α1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated α1-antitrypsin in the ER. In hepatoma cells, shift of mutated α1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated α1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.

scholarly journals The unconventional biogenesis of Kv7.1-KCNE1 complexes

2020 ◽  
Vol 6 (14) ◽  
pp. eaay4472 ◽  
Author(s):  
Anna Oliveras ◽  
Clara Serrano-Novillo ◽  
Cristina Moreno ◽  
Alicia de la Cruz ◽  
Carmen Valenzuela ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Regulatory Subunit ◽  
Long Qt ◽  
Ongoing Debate ◽  
Subcellular Compartment ◽  
Ongoing Controversy ◽  
The Subject ◽  
Complex Forms

The potassium channel Kv7.1 associates with the KCNE1 regulatory subunit to trigger cardiac IKs currents. Although the Kv7.1/KCNE1 complex has received much attention, the subcellular compartment hosting the assembly is the subject of ongoing debate. Evidence suggests that the complex forms either earlier in the endoplasmic reticulum or directly at the plasma membrane. Kv7.1 and KCNE1 mutations, responsible for long QT syndromes, impair association and traffic, thereby altering IKs currents. We found that Kv7.1 and KCNE1 do not assemble in the first stages of their biogenesis. Data support an unconventional secretory pathway for Kv7.1-KCNE1 that bypasses Golgi. This route targets channels to endoplasmic reticulum–plasma membrane junctions, where Kv7.1-KCNE1 assemble. This mechanism helps to resolve the ongoing controversy about the subcellular compartment hosting the association. Our results also provide new insights into IKs channel localization at endoplasmic reticulum–plasma membrane junctions, highlighting an alternative anterograde trafficking mechanism for oligomeric ion channels.

scholarly journals Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

2008 ◽  
Vol 8 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Connie B. Nichols ◽  
Jessica Ferreyra ◽  
Elizabeth R. Ballou ◽  
J. Andrew Alspaugh
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Cryptococcus Neoformans ◽  
Sexual Differentiation ◽  
Signaling Cascades ◽  
Ras Signaling ◽  
Signaling Specificity ◽  
Downstream Signaling ◽  
Functional Flexibility ◽  
Human Fungal Pathogen

ABSTRACT In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.

scholarly journals Role of p97 and Syntaxin 5 in the Assembly of Transitional Endoplasmic Reticulum

2000 ◽  
Vol 11 (8) ◽  
pp. 2529-2542 ◽  
Author(s):  
Line Roy ◽  
John J.M. Bergeron ◽  
Christine Lavoie ◽  
Rob Hendriks ◽  
Jennifer Gushue ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Atp Hydrolysis ◽  
Smooth Endoplasmic Reticulum ◽  
Atp Depletion ◽  
Low Density ◽  
Transitional Endoplasmic Reticulum ◽  
Membrane Compartment ◽  
A Cell

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc− 1) isolated from rat liver homogenates reconstitute tER by Mg2+GTP- and Mg2+ATP-hydrolysis–dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.

scholarly journals Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

1986 ◽  
Vol 103 (4) ◽  
pp. 1179-1191 ◽  
Author(s):  
C S Copeland ◽  
R W Doms ◽  
E M Bolzau ◽  
R G Webster ◽  
A Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Subunit Interactions ◽  
Oligomer Formation ◽  
Influenza Hemagglutinin ◽  
Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

2007 ◽  
Vol 409 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Satoshi Takida ◽  
Yusuke Maeda ◽  
Taroh Kinoshita
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Direct Evidence ◽  
Temperature Sensitive ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
P24 Proteins

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

scholarly journals Transmembrane domain–dependent partitioning of membrane proteins within the endoplasmic reticulum

2008 ◽  
Vol 181 (1) ◽  
pp. 105-118 ◽  
Author(s):  
Paolo Ronchi ◽  
Sara Colombo ◽  
Maura Francolini ◽  
Nica Borgese
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Fluorescent Proteins ◽  
Lipid Interactions ◽  
Er Exit Sites ◽  
Physicochemical Features ◽  
Er Membrane

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein–protein and protein–lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail–anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER–Golgi boundary by simple receptor-independent mechanisms based on partitioning.

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