scholarly journals The endophilin curvature-sensitive motif requires electrostatic guidance to recycle synaptic vesicles in vivo

2021 ◽  
Author(s):  
Lin Zhang ◽  
Yu Wang ◽  
Yongming Dong ◽  
Aaradhya Pant ◽  
Yan Liu ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Nuclear Pore ◽  
Functional Specificity ◽  
Curvature Sensing ◽  
Vesicle Recycling ◽  
Endocytic Protein ◽  
Complex Protein ◽  
Common Deficiency

Curvature-sensing mechanisms assist proteins in executing particular actions on various membrane organelles. Here, we investigated the functional specificity of curvature-sensing amphipathic motifs through the study of endophilin, an endocytic protein for synaptic vesicle recycling. We generated chimeric endophilin proteins by replacing the endophilin amphipathic motif H0 with other curvature-sensing amphipathic motifs. We found that the role of amphipathic motifs cannot simply be extrapolated from the identity of their parental proteins. For example, the amphipathic motif of the nuclear pore complex protein NUP133 functionally replaced the synaptic role of endophilin H0. Interestingly, non-functional endophilin chimeras had similar defects — producing fewer synaptic vesicles but more endosomes — indicating that the curvature-sensing motifs in these chimeras have a common deficiency at reforming synaptic vesicles. Finally, we converted non-functional endophilin chimeras into functional proteins by changing the cationic property of amphipathic motifs, setting a precedent for reprogramming the functional specificity of curvature-sensing motifs in vivo.

scholarly journals Dual pools of actin at presynaptic terminals

2012 ◽  
Vol 107 (12) ◽  
pp. 3479-3492 ◽  
Author(s):  
Adam Bleckert ◽  
Huzefa Photowala ◽  
Simon Alford
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Repetitive Stimulation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Vesicle Recycling ◽  
Latrunculin A ◽  
Kinetics Of

We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals Extracellular Signal-Regulated Kinase 2 (ERK2) Phosphorylation Sites and Docking Domain on the Nuclear Pore Complex Protein Tpr Cooperatively Regulate ERK2-Tpr Interaction

2008 ◽  
Vol 28 (22) ◽  
pp. 6954-6966 ◽  
Author(s):  
Tomáš Vomastek ◽  
Marcin P. Iwanicki ◽  
W. Richard Burack ◽  
Divya Tiwari ◽  
Devanand Kumar ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Ectopic Expression ◽  
Nuclear Pore ◽  
Phosphorylation Sites ◽  
Biological Responses ◽  
Extracellular Signal ◽  
Complex Protein ◽  
Mitogen Activated Protein ◽  
Bona Fide

ABSTRACT Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

scholarly journals Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

1996 ◽  
Vol 7 (6) ◽  
pp. 917-934 ◽  
Author(s):  
A L Goldstein ◽  
C A Snay ◽  
C V Heath ◽  
C N Cole
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Main Body ◽  
The Novel ◽  
Conditional Allele ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Export ◽  
In Cells

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

scholarly journals Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs

2005 ◽  
Vol 168 (7) ◽  
pp. 1013-1025 ◽  
Author(s):  
Eva Kiesler ◽  
Manuela E. Hase ◽  
David Brodin ◽  
Neus Visa
Keyword(s):  
Rna Binding ◽  
M Protein ◽  
Nuclear Pore ◽  
Binding Domains ◽  
Mrna Targets ◽  
Modular Domain ◽  
Exonic Splicing Enhancers ◽  
Rna Complexes

Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59–RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

scholarly journals ZC3HC1 is a structural element of the nuclear basket effecting interlinkage of TPR polypeptides

2022 ◽  
Author(s):  
Philip Gunkel ◽  
Volker C Cordes
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Structural Element ◽  
Building Element ◽  
Rapid Elimination

The nuclear basket (NB), anchored to the nuclear pore complex (NPC), is commonly thought of as built solely of protein TPR polypeptides, the latter thus regarded as the NB's only scaffold-forming components. In the current study, we report ZC3HC1 as a second building element of the NB. Recently described as an NB-appended protein omnipresent in vertebrates, we now show that ZC3HC1, both in vivo and in vitro, enables in a step-wise fashion the recruitment of TPR subpopulations to the NB and their linkage to already NPC-anchored TPR polypeptides. We further demonstrate that the degron-mediated rapid elimination of ZC3HC1 results in the prompt detachment of the ZC3HC1-appended TPR polypeptides from the NB and their release back into the nucleoplasm again, underscoring the role of ZC3HC1 as a natural structural element of the NB. Finally, we show that ZC3HC1 can keep TPR polypeptides positioned even at sites remote from the NB, in line with ZC3HC1 functioning as a protein connecting TPR polypeptides. 

scholarly journals A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo.

1988 ◽  
Vol 107 (4) ◽  
pp. 1289-1297 ◽  
Author(s):  
C Featherstone ◽  
M K Darby ◽  
L Gerace
Keyword(s):  
Monoclonal Antibody ◽  
Ribosomal Rna ◽  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Small Proteins

A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport.

scholarly journals Sec13 Shuttles between the Nucleus and the Cytoplasm and Stably Interacts with Nup96 at the Nuclear Pore Complex

2003 ◽  
Vol 23 (20) ◽  
pp. 7271-7284 ◽  
Author(s):  
Jost Enninga ◽  
Agata Levay ◽  
Beatriz M. A. Fontoura
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Yeast Two Hybrid ◽  
Biochemical Assays ◽  
Spindle Apparatus ◽  
Complex Protein ◽  
Localization Signal ◽  
Two Hybrid

ABSTRACT Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

scholarly journals Interactions between non-structured domains of FG- and non-FG-nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis

2018 ◽  
Author(s):  
Hide A. Konishi ◽  
Shige H. Yoshimura
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Middle Stage ◽  
Selective Channel ◽  
Sequential Addition ◽  
Ordered Assembly ◽  
Self Assembled

SummaryIn this study, we examined how channel-forming subunits of the nuclear pore complex (NPC) are assembled into a selective channel within a highly structured scaffold ring during post-mitotic assembly. We focused on non-structured domains of the scaffold Nups and performed in vitro self-assembled particle assays with those derived from channel-forming FG-Nups. We found that non-structured domains of ELYS and Nup35N interacted with channel-forming FG-Nups to form a self-assembled particle. Sequential addition of FG-Nups into the scaffold particle revealed that ELYS, which initiates post-mitotic NPC reassembly, interacts with early assembling FG-Nups (Nups98 and 153) but not middle stage-assembling FG-Nups (Nups58 and 62). Nup35, which assembles between the early and middle stages, facilitated the assembly of Nup62 into the early assembling Nups both in vitro and in vivo. These results demonstrate that ELYS and Nup35 have a role of facilitator in the ordered assembly of channel-forming FG-Nups during mitosis.

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