Regulation of neuronal progenitor delamination by dynein-driven post-Golgi apical transport

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that this delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3, and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knock-out of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.

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Activation of the Kexin from Schizosaccharomyces pombeRequires Internal Cleavage of Its Initially Cleaved Prosequence

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.400 ◽

1998 ◽

Vol 18 (1) ◽

pp. 400-408 ◽

Cited By ~ 21

Author(s):

Dale Powner ◽

John Davey

Keyword(s):

Cleavage Site ◽

Secretory Pathway ◽

Catalytic Domain ◽

Activation Process ◽

Processing Enzymes ◽

Free Translation ◽

A Cell ◽

Internal Site ◽

Primary Cleavage ◽

Full Activation

ABSTRACT Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeastSchizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopuseggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102↓ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombecells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.

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Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells

Journal of Cell Science ◽

10.1242/jcs.106.3.731 ◽

1993 ◽

Vol 106 (3) ◽

pp. 731-740 ◽

Cited By ~ 2

Author(s):

E.S. Schweitzer

Keyword(s):

Pc12 Cells ◽

Secretory Pathway ◽

Intracellular Localization ◽

Synaptic Function ◽

Molecular Forms ◽

Secretory Proteins ◽

Secretory Pathways ◽

Regulated Secretion ◽

Constitutive Secretion ◽

A Cell

PC12 cells secrete the enzyme acetylcholinesterase (AChE) while at rest, and increase the overall rate of this secretion 2-fold upon depolarization. This behavior is different from the release of other markers by the constitutive or regulated secretory pathways in PC12 cells. Both the resting and stimulated release of AChE are unchanged after treatment with a membrane-impermeable esterase inhibitor, demonstrating that it represents true secretion and not shedding from the cell surface. The stimulation release of AChE is Ca(2+)-dependent, while the unstimulated release is not. Analysis of the molecular forms of AChE secreted by PC12 cells indicates that the release of AChE actually involves two concurrent but independent secretory processes, and that the G4 form of the enzyme is secreted constitutively, while both the G2 and G4 forms are secreted in a regulated manner, presumably from regulated secretory vesicles. Compared with other regulated secretory proteins, a much smaller fraction of cellular AChE is secreted, and the intracellular localization of this enzyme differs from that of other regulated secretory proteins. The demonstration that a cell line that exhibits regulated secretion of acetylcholine (ACh) is also capable of regulated secretion of AChE provides additional evidence for the existence of multiple regulated secretory pathways within a single cell. Moreover, there appears to be a selective packaging of different molecular forms of AChE into the regulated versus the constitutive secretory pathway. Both the specificity of sorting of AChE and the regulation of its secretion suggest that AChE may play a more dynamic role in synaptic function than has been recognized previously.

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Secretion of Cryparin, a Fungal Hydrophobin

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5431-5435.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5431-5435 ◽

Cited By ~ 15

Author(s):

Patricia M. McCabe ◽

Neal K. Van Alfen

Keyword(s):

Cell Wall ◽

Cell Walls ◽

Submerged Culture ◽

Secretory Pathway ◽

Culture Medium ◽

Secretory Vesicle ◽

Cryphonectria Parasitica ◽

Secreted Protein ◽

Filamentous Ascomycete ◽

A Cell

ABSTRACT Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls ofC. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.

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Mechanical bistability enabled by ectodermal compression facilitates Drosophila mesoderm invagination

10.1101/2021.03.18.435928 ◽

2021 ◽

Author(s):

Hanqing Guo ◽

Michael Swan ◽

Shicheng Huang ◽

Bing He

Keyword(s):

Myosin Ii ◽

Cell Sheet ◽

Apical Surface ◽

Apical Constriction ◽

Out Of Plane ◽

A Cell ◽

Plane Compression ◽

Contractile Forces ◽

Tissue Relaxation ◽

Muscle Myosin

Apical constriction driven by non-muscle myosin II (″myosin″) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of myosin, we find that during Drosophila mesoderm invagination, myosin contractility is critical to prevent tissue relaxation during the early, ″priming″ stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration, suggesting that the mesoderm is mechanically bistable during gastrulation. Combining computer modeling and experimental measurements, we show that the observed mechanical bistability arises from an in-plane compression from the surrounding ectoderm, which promotes mesoderm invagination by facilitating a buckling transition. Our results indicate that Drosophila mesoderm invagination requires a joint action of local apical constriction and global in-plane compression to trigger epithelial buckling.

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Spa33, a Cell Surface-Associated Subunit of the Mxi-Spa Type III Secretory Pathway of Shigella flexneri, Regulates Ipa Protein Traffic

Infection and Immunity ◽

10.1128/iai.69.4.2180-2189.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2180-2189 ◽

Cited By ~ 24

Author(s):

Raymond Schuch ◽

Anthony T. Maurelli

Keyword(s):

Cell Surface ◽

Secretory Pathway ◽

Shigella Flexneri ◽

Mobile Element ◽

Cell Contact ◽

Protein Traffic ◽

Type Iii ◽

Transmembrane Channel ◽

A Cell ◽

Spa Type

ABSTRACT The Mxi-Spa type III secretion system of Shigella flexneri directs the host cell contact-induced secretion of a set of invasins, referred to as Ipas. In this study, we examined the role of Spa33 in Ipa secretion. A spa33-null mutant was both noninvasive and unable to translocate the Ipas from inner membrane to outer membrane (OM) positions of the Mxi-Spa transmembrane channel. Spa33 was found to be a Mxi-Spa substrate that is translocated to the bacterial cell surface upon the induction of Ipa secretion. This mobility may serve to drive Ipa translocation within Mxi-Spa toward OM positions. Consistent with a second distinct role in regulating Ipa traffic, the overexpression of Spa33 also blocked Ipa secretion and resulted in Ipa accumulation at the OM. Co-overexpression of Spa33 and another OM-associated element, Spa32, did not disrupt Ipa secretion, suggesting an interaction between the two proteins and an effect on the mechanism which serves to regulate Ipa release from the OM. These findings indicate that Spa33 is a mobile element within Mxi-Spa, which is required to control Ipa translocation into and out of OM positions of the secretory structure.

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Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast

Journal of Cell Science ◽

10.1242/jcs.115.2.421 ◽

2002 ◽

Vol 115 (2) ◽

pp. 421-431

Author(s):

Anna Matynia ◽

Sandra S. Salus ◽

Shelley Sazer

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Fission Yeast ◽

Cell Cycle Progression ◽

Secretory Pathway ◽

Yeast Cells ◽

Ran Gtpase ◽

A Cell ◽

Cell Cycle Block ◽

Er Membrane

The Ran GTPase is an essential protein that has multiple functions in eukaryotic cells. Fission yeast cells in which Ran is misregulated arrest after mitosis with condensed, unreplicated chromosomes and abnormal nuclear envelopes. The fission yeast sns mutants arrest with a similar cell cycle block and interact genetically with the Ran system. sns-A10, sns-B2 and sns-B9 have mutations in the fission yeast homologues of S. cerevisiae Sar1p, Sec31p and Sec53p, respectively, which are required for the early steps of the protein secretory pathway. The three sns mutants accumulate a normally secreted protein in the endoplasmic reticulum (ER), have an increased amount of ER membrane, and the ER/nuclear envelope lumen is dilated. Neither a post-ER block in the secretory pathway, nor ER proliferation caused by overexpression of an integral ER membrane protein, results in a cell cycle-specific defect. Therefore, the arrest seen in sns-A10, sns-B2 and sns-B9 is most likely due to nuclear envelope defects that render the cells unable to re-establish the interphase organization of the nucleus after mitosis. As a consequence, these mutants are unable to decondense their chromosomes or to initiate of the next round of DNA replication.

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Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction.

The Journal of Cell Biology ◽

10.1083/jcb.118.5.1057 ◽

1992 ◽

Vol 118 (5) ◽

pp. 1057-1070 ◽

Cited By ~ 12

Author(s):

B A Lee ◽

D J Donoghue

Keyword(s):

Cell Surface ◽

Secretory Pathway ◽

Transmembrane Protein ◽

Immunofluorescent Localization ◽

Receptor Ligand ◽

Surface Transport ◽

Established Cell ◽

Autocrine Transformation ◽

Nih 3T3 ◽

Retention Signal

An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membrane-anchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to post-Golgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not down-regulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0587 ◽

2008 ◽

Vol 19 (2) ◽

pp. 572-586 ◽

Cited By ~ 48

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.

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Spatial distribution and characterization of non-apical progenitors in the zebrafish embryo central nervous system

Open Biology ◽

10.1098/rsob.160312 ◽

2017 ◽

Vol 7 (2) ◽

pp. 160312 ◽

Cited By ~ 16

Author(s):

Rebecca McIntosh ◽

Joseph Norris ◽

Jon D. Clarke ◽

Paula Alexandre

Keyword(s):

Spinal Cord ◽

Central Nervous System ◽

Nervous System ◽

Mammalian Species ◽

Small Population ◽

Mammalian Brain ◽

Molecular Characteristics ◽

Apical Surface ◽

Brain Expansion

Studies of non-apical progenitors (NAPs) have been largely limited to the developing mammalian cortex. They are postulated to generate the increase in neuron numbers that underlie mammalian brain expansion. Recently, NAPs have also been reported in the retina and central nervous system of non-mammalian species; in the latter, however, they remain poorly characterized. Here, we characterize NAP location along the zebrafish central nervous system during embryonic development, and determine their cellular and molecular characteristics and renewal capacity. We identified a small population of NAPs in the spinal cord, hindbrain and telencephalon of zebrafish embryos. Live-imaging analysis revealed at least two types of mitotic behaviour in the telencephalon: one NAP subtype retains the apical attachment during division, while another divides in a subapical position disconnected from the apical surface. All NAPs observed in spinal cord lost apical contact prior to mitoses. These NAPs express HuC and produce two neurons from a single division. Manipulation of Notch activity reveals that neurons and NAPs in the spinal cord use similar regulatory mechanisms. This work suggests that the majority of spinal NAPs in zebrafish share characteristics with basal progenitors in mammalian brains.

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Codon bias determines sorting of ion channel protein

10.1101/2020.03.17.994780 ◽

2020 ◽

Author(s):

Marina Kithil ◽

Anja Jeannine Engel ◽

Markus Langhans ◽

Oliver Rauh ◽

Matea Cartolano ◽

...

Keyword(s):

Mammalian Cells ◽

Secretory Pathway ◽

Model Systems ◽

Dependent Manner ◽

Intracellular Proteins ◽

Polypeptide Folding ◽

A Cell ◽

Identical Amino Acid Sequence ◽

Cycle Dependent ◽

The Impact

AbstractThe choice of codons can influence local translation kinetics during protein synthesis. The question of whether the modulation of polypeptide folding and binding to chaperons influences sorting of nascent membrane proteins remains unclear. Here, we use two similar K+ channels as model systems to examine the impact of codon choice on protein sorting. By monitoring transient expression of GFP tagged proteins in mammalian cells we find that targeting of one channel to the secretory pathway is insensitive to codon optimization. In contrast, sorting of the second channel to the mitochondria is very sensitive to codon choice. The protein with an identical amino acid sequence is sorted in a codon and cell cycle dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves together with a cell-state depending decoding mechanism as a secondary code for sorting intracellular proteins.

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