scholarly journals The luminal AAA+ ATPase torsinA mediates distinct mechanisms of nuclear-cytoplasmic communication by adopting different functional assembly states.

2021 ◽  
Author(s):  
Kwang-Ho Hur ◽  
Jared W. Hennen ◽  
Cosmo A Saunders ◽  
Amy Schoenhoefen ◽  
Patrick T Willey ◽  
...  
Keyword(s):  
Nuclear Pore ◽  
Linc Complex ◽  
Complex Assembly ◽  
Aaa Atpase ◽  
Cellular Assays ◽  
Dyt1 Dystonia ◽  
Helical Polymer ◽  
Aaa Protein

Chemical and mechanical nuclear-cytoplasmic communication across the nuclear envelope (NE) is largely mediated by the nuclear pore complex (NPC) and the linker of nucleoskeleton and cytoskeleton (LINC) complex, respectively. While NPC and LINC complex assembly are functionally related, the mechanisms responsible for this relationship remain poorly understood. Here, we investigated how the luminal ATPases associated with various cellular activities (AAA+) protein torsinA promotes NPC and LINC complex assembly using fluorescence fluctuation spectroscopy (FFS), quantitative photobleaching analyses, and functional cellular assays. We report that torsinA controls LINC complex-dependent nuclear-cytoskeletal coupling as a soluble hexameric AAA+ protein and interphase NPC biogenesis as a membrane-associated helical polymer. These findings help resolve the conflicting models of torsinA function that were recently proposed based on in vitro structural studies. Our results will enable future studies of the role of defective nuclear-cytoplasmic communication in DYT1 dystonia and other diseases caused by mutations in torsinA.

scholarly journals MLF2 modulates phase separated nuclear envelope condensates that provoke dual proteotoxicity

2021 ◽  
Author(s):  
Sarah M Prophet ◽  
Anthony J Rampello ◽  
Robert F Niescier ◽  
Juliana E Shaw ◽  
Anthony J Koleske ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Envelope ◽  
Nuclear Pore ◽  
Disease Manifestation ◽  
Disease Etiology ◽  
Aaa Atpase ◽  
Dyt1 Dystonia ◽  
Chaperone Network ◽  
Neurological Movement Disorder

DYT1 dystonia is a highly debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is presently unknown. We report that FG-nucleoporins in the bleb lumen undergo phase separation and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitinated proteins that are normally rapidly degraded in healthy cells partition into the bleb lumen and become stabilized. Additionally, blebs selectively sequester a chaperone network composed of HSP70s and HSP40s. The composition of this chaperone network is altered by the bleb component MLF2. We further demonstrate that MLF2 is a catalyst of phase separation that suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our studies identify unprecedented, dual mechanisms of proteotoxicity in the context of liquid-liquid phase separation with direct implications for our understanding of disease etiology and treatment.

scholarly journals DYT1 dystonia patient-derived fibroblasts have increased deformability and susceptibility to damage by mechanical forces

2018 ◽  
Author(s):  
Navjot Kaur Gill ◽  
Chau Ly ◽  
Paul H. Kim ◽  
Cosmo A. Saunders ◽  
Loren G. Fong ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Mechanical Stretch ◽  
Mechanical Forces ◽  
Linc Complex ◽  
Loss Of Function ◽  
Mouse Fibroblasts ◽  
Dyt1 Dystonia ◽  
Sense And Respond ◽  
Aaa Protein

AbstractDYT1 dystonia is a neurological movement disorder that is caused by a loss-of-function mutation in the DYT1/TOR1A gene, which encodes torsinA, the luminal ATPase-associated (AAA+) protein. TorsinA is required for the assembly of functional linker of nucleoskeleton and cytoskeleton (LINC) complexes, and consequently the mechanical integration of the nucleus and the cytoskeleton. Despite the potential implications of altered mechanobiology in dystonia pathogenesis, the role of torsinA in regulating cellular mechanical phenotype, or mechanotype, in DYT1 dystonia remains unknown. Here, we define the mechanotype of mouse fibroblasts lacking functional torsinA as well as human fibroblasts isolated from DYT1 dystonia patients. We find that the deletion of torsinA or the expression of torsinA containing the DYT1 dystonia-causing ΔE302/303 (ΔE) mutation results in a more deformable cellular mechanotype. We observe a similar increased deformability of mouse fibroblasts that lack lamina-associated polypeptide 1 (LAP1), which interacts with and stimulates the ATPase activity of torsinA in vitro; as well as with depletion of the LINC complex proteins, Sad1/UNC-84 (SUN)1 and SUN2, lamin A/C, or lamin B1. Moreover, we report that DYT1 dystonia patient-derived fibroblasts are more compliant than fibroblasts isolated from unafflicted individuals. DYT1 fibroblasts also exhibit increased nuclear strain and decreased viability following mechanical stretch. Taken together, our results support a model where the physical connectivity between the cytoskeleton and nucleus contributes to cellular mechanotype. These findings establish the foundation for future mechanistic studies to understand how altered cellular mechanotype may contribute to DYT1 dystonia pathogenesis; this may be particularly relevant in the context of how neurons sense and respond to mechanical forces during traumatic brain injury, which is known to be a major cause of acquired dystonia.

Cellular Samurai: katanin and the severing of microtubules

2000 ◽  
Vol 113 (16) ◽  
pp. 2821-2827 ◽  
Author(s):  
L. Quarmby
Keyword(s):  
Epithelial Cells ◽  
Essential Role ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Biochemical Studies ◽  
New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

scholarly journals A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G.W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Experimental System ◽  
Free Expression ◽  
Supported Lipid Bilayers ◽  
Linc Complex ◽  
Complex Assembly ◽  
Complex Core

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

scholarly journals Expression of TorsinA in a heterologous yeast system reveals interactions with conserved lumenal domains of LINC and nuclear pore complexes

2018 ◽  
Author(s):  
Madeleine Chalfant ◽  
Karl W. Barber ◽  
Sapan Borah ◽  
David Thaller ◽  
C. Patrick Lusk
Keyword(s):  
Nuclear Envelope ◽  
Genetic Interaction ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Physical Interaction ◽  
Nuclear Pore Complexes ◽  
Gene Encoding ◽  
Aaa Atpase ◽  
Dyt1 Dystonia ◽  
Pore Complexes

ABSTRACTDYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA. TorsinA localizes within the lumen of the nuclear envelope/ER and binds to a membrane-spanning co-factor, LAP1 or LULL1, to form an ATPase; the substrate(s) of TorsinA remain ill defined. Here we use budding yeast, which lack Torsins, to interrogate TorsinA function. We show that TorsinA accumulates at nuclear envelope embedded spindle pole bodies (SPBs) in a way that requires its oligomerization and the conserved SUN-domain protein, Mps3. TorsinA is released from SPBs upon expression of LAP1 and stabilized by LAP1 mutants incapable of stimulating TorsinA ATPase activity, suggesting the recapitulation of a TorsinA-substrate cycle. While the expression of TorsinA or TorsinA-ΔE impacts the fitness of strains expressing mps3 alleles, a genetic interaction with a conserved component of the nuclear pore complex, Pom152, is specific for TorsinA. This specificity is mirrored by a physical interaction between Pom152 and TorsinA, but not TorsinA-ΔE. These data suggest that TorsinA-nucleoporin interactions would be abrogated by TorsinA-ΔE, providing new experimental avenues to interrogate the molecular basis behind nuclear envelope herniations seen in cells lacking TorsinA function.

scholarly journals Decreased mechanotransduction prevents nuclear collapse in aCaenorhabditis eleganslaminopathy

2020 ◽  
Vol 117 (49) ◽  
pp. 31301-31308
Author(s):  
Gabriela Huelgas-Morales ◽  
Mark Sanders ◽  
Gemechu Mekonnen ◽  
Tatsuya Tsukamoto ◽  
David Greenstein
Keyword(s):  
Nuclear Envelope ◽  
Complex Function ◽  
Nuclear Lamina ◽  
Premature Aging ◽  
Nuclear Pore ◽  
Linc Complex ◽  
Force Transmission ◽  
Cell Nuclei ◽  
Aaa Atpase ◽  
C Elegans

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use theCaenorhabditis elegansgermline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis—processes that require LINC complex function. We report that decreasing the function of theC. eleganstorsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA–LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

scholarly journals The Role of Xgrip210 in γ-Tubulin Ring Complex Assembly and Centrosome Recruitment

2000 ◽  
Vol 151 (7) ◽  
pp. 1525-1536 ◽  
Author(s):  
Lijun Zhang ◽  
Thomas J. Keating ◽  
Andrew Wilde ◽  
Gary G. Borisy ◽  
Yixian Zheng
Keyword(s):  
Immunogold Labeling ◽  
Structural Studies ◽  
Complex Assembly ◽  
Interacting Protein ◽  
Centrosomal Protein ◽  
Ring Complex

The γ-tubulin ring complex (γTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that γTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the γTuRC. Consequently, the cap structure of the γTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. Here, we report the cloning and characterization of a new subunit of Xenopus γTuRC, Xgrip210. We show that Xgrip210 is a conserved centrosomal protein that is essential for the formation of γTuRC. Using immunogold labeling, we found that Xgrip210 is localized to the ends of microtubules nucleated by the γTuRC and that its localization is more distal, toward the tip of the γTuRC-cap structure, than that of γ-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the γTuRC, but also the recruitment of γ-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the γTuRC cap structure that is required for the assembly of the γTuRC.

Nitric oxide inhibits the ATPase activity of the chaperone-like AAA+ ATPase CDC48, a target for S-nitrosylation in cryptogein signalling in tobacco cells

2012 ◽  
Vol 447 (2) ◽  
pp. 249-260 ◽  
Author(s):  
Jéremy Astier ◽  
Angélique Besson-Bard ◽  
Olivier Lamotte ◽  
Jean Bertoldo ◽  
Stéphane Bourque ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Oxidative Modification ◽  
Tobacco Cells ◽  
No Donors ◽  
Defence Reaction ◽  
Aaa Atpase ◽  
Proteomic Approach ◽  
D2 Domain

NO has important physiological functions in plants, including the adaptative response to pathogen attack. We previously demonstrated that cryptogein, an elicitor of defence reaction produced by the oomycete Phytophthora cryptogea, triggers NO synthesis in tobacco. To decipher the role of NO in tobacco cells elicited by cryptogein, in the present study we performed a proteomic approach in order to identify proteins undergoing S-nitrosylation. We provided evidence that cryptogein induced the S-nitrosylation of several proteins and identified 11 candidates, including CDC48 (cell division cycle 48), a member of the AAA+ ATPase (ATPase associated with various cellular activities) family. In vitro, NtCDC48 (Nicotiana tabacum CDC48) was shown to be poly-S-nitrosylated by NO donors and we could identify Cys110, Cys526 and Cys664 as a targets for S-nitrosylation. Cys526 is located in the Walker A motif of the D2 domain, that is involved in ATP binding and was previously reported to be regulated by oxidative modification in Drosophila. We investigated the consequence of NtCDC48 S-nitrosylation and found that NO abolished NtCDC48 ATPase activity and induced slight conformation changes in the vicinity of Cys526. Similarly, substitution of Cys526 by an alanine residue had an impact on NtCDC48 activity. More generally, the present study identified CDC48 as a new candidate for S-nitrosylation in plants facing biotic stress and further supports the importance of Cys526 in the regulation of CDC48 by oxidative/nitrosative agents.

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 The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis

2015 ◽  
Vol 208 (6) ◽  
pp. 671-681 ◽  
Author(s):  
J. Sebastian Gomez-Cavazos ◽  
Martin W. Hetzer
Keyword(s):  
Cell Differentiation ◽  
Nuclear Envelope ◽  
Er Stress ◽  
Myoblast Differentiation ◽  
Nuclear Pore ◽  
C2c12 Myoblast ◽  
Terminal Domain ◽  
Er Homeostasis

Previously, we identified the nucleoporin gp210/Nup210 as a critical regulator of muscle and neuronal differentiation, but how this nucleoporin exerts its function and whether it modulates nuclear pore complex (NPC) activity remain unknown. Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space. Removal of the C-terminal domain, which partially mislocalizes gp210/Nup210 away from NPCs, efficiently rescues the differentiation defect caused by the knockdown of endogenous gp210/Nup210. Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation. We demonstrate that the endoplasmic reticulum (ER) stress-specific caspase cascade is exacerbated during Nup210 depletion and that blocking ER stress-mediated apoptosis rescues differentiation of Nup210-deficient cells. Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis.

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