Phosphorylation of the microtubule-severing AAA+ enzyme Katanin regulates C. elegans embryo development

The evolutionarily conserved microtubule (MT)-severing AAA-ATPase enzyme Katanin is emerging as a critical regulator of MT dynamics. In Caenorhabditis elegans, Katanin MT-severing activity is essential for meiotic spindle assembly but is toxic for the mitotic spindle. Here we analyzed Katanin dynamics in C. elegans and deciphered the role of Katanin phosphorylation in the regulation of its activity and stability. Katanin is abundant in oocytes, and its levels drop after meiosis, but unexpectedly, a significant fraction is present throughout embryogenesis, where it is dynamically recruited to the centrosomes and chromosomes during mitosis. We show that the minibrain kinase MBK-2, which is activated during meiosis, phosphorylates Katanin at multiple serines. We demonstrate unequivocally that Katanin phosphorylation at a single residue is necessary and sufficient to target Katanin for proteasomal degradation after meiosis, whereas phosphorylation at the other sites only inhibits Katanin ATPase activity stimulated by MTs. Our findings suggest that cycles of phosphorylation and dephosphorylation fine-tune Katanin level and activity to deliver the appropriate MT-severing activity during development.

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MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis

Genes & Development ◽

10.1101/gad.14.9.1072 ◽

2000 ◽

Vol 14 (9) ◽

pp. 1072-1084

Author(s):

Martin Srayko ◽

Dan W. Buster ◽

Omar A. Bazirgan ◽

Francis J. McNally ◽

Paul E. Mains

Keyword(s):

Caenorhabditis Elegans ◽

Hela Cells ◽

Mitotic Spindle ◽

Subcellular Location ◽

Meiotic Spindle ◽

Aaa Atpase ◽

C Elegans ◽

Microtubule Severing ◽

Acid Protein ◽

Spindle Function

The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm ∼20 minutes apart. Themei-1 and mei-2 genes of C. elegans are required for the establishment of the oocyte meiotic spindle but are not required for mitotic spindle function. mei-1 encodes an AAA ATPase family member with similarity to the p60 catalytic subunit of the heterodimeric sea urchin microtubule-severing protein, katanin. We report that mei-2 encodes a 280-amino acid protein containing a region similar to the p80-targeting subunit of katanin. MEI-1 and MEI-2 antibodies decorate the polar ends of meiotic spindle microtubules and meiotic chromatin. We find that the subcellular location of MEI-2 depends on wild-type mei-1 activity and vice versa. These experiments, combined with MEI-1 and MEI-2's similarity to p60 and p80 katanin, suggest that the C. elegans proteins function as a complex. In support of this idea, MEI-1 and MEI-2 physically associate in HeLa cells. Furthermore, co-expression of MEI-1 and MEI-2 in HeLa cells results in the disassembly of microtubules. These data lead us to conclude that MEI-1/MEI-2 microtubule-severing activity is required for meiotic spindle organization in C. elegans.

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Cellular Samurai: katanin and the severing of microtubules

Journal of Cell Science ◽

10.1242/jcs.113.16.2821 ◽

2000 ◽

Vol 113 (16) ◽

pp. 2821-2827 ◽

Cited By ~ 5

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.

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WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium

10.21203/rs.3.rs-622797/v1 ◽

2021 ◽

Author(s):

Sebiha Cevik ◽

Lama Alabdi ◽

Xiaoyu Peng ◽

Tina Beyer ◽

Atiyye Zorluer ◽

...

Keyword(s):

Clinical Features ◽

Renal Agenesis ◽

Rare Disorders ◽

Compartment Analysis ◽

C Elegans ◽

Gtpase Activating Proteins ◽

Mechanistic Analysis ◽

Evolutionarily Conserved ◽

Null Mutants

Abstract The term “ciliopathy” refers to a group of over 35 rare disorders characterized by defective cilia and many overlapping clinical features, such as hydrocephalus, cerebellar vermis hypoplasia, polydactyly, and retinopathy. Even though many genes have been implicated in ciliopathies, the genetic pathogenesis in certain cases remains still undisclosed. Here, we identified a homozygous truncating variant in WDR31 in a patient with a typical ciliopathy phenotype encompassing congenital hydrocephalus, polydactyly, and renal agenesis. WDR31 is an evolutionarily conserved protein that localizes to the cilium and cilia-related compartment. Analysis from zebrafish supports the role of WDR31 in regulating the cilia morphology. The CRISPR/Cas9 knock-in (p.Arg261del) C. elegans model of the patient variant (p.Arg268*) reproduced several cilia-related defects observed in wdr-31 null mutants. Mechanistic analysis from C. elegans revealed that WDR-31 functions redundantly with ELDM-1 (ELMOD protein) and RPI-2 (RP2) to regulate the IFT trafficking through controlling the cilia entry of the BBSome. This work revealed WDR31 as a new ciliopathy protein that regulates IFT and BBSome trafficking.

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A New Trick for an Old Dogma: ENaC Proteins as Mechanotransducers in Vascular Smooth Muscle

Physiology ◽

10.1152/physiol.00034.2007 ◽

2008 ◽

Vol 23 (1) ◽

pp. 23-31 ◽

Cited By ~ 63

Author(s):

Heather A. Drummond ◽

Samira C. Grifoni ◽

Nikki L. Jernigan

Keyword(s):

Extracellular Matrix ◽

Cytoskeletal Proteins ◽

Protective Mechanism ◽

C Elegans ◽

Mechanical Signaling ◽

Evolutionarily Conserved ◽

Ion Conducting ◽

New Perspective ◽

Renal Blood Flow Autoregulation

Myogenic constriction is a vasoconstriction of blood vessels to increases in perfusion pressure. In renal preglomerular vasculature, it is an established mechanism of renal blood flow autoregulation. Recently, myogenic constriction has been identified as an important protective mechanism, preventing the transmission of systemic pressure to the fragile glomerular vasculature. Although the signal transduction pathways mediating vasoconstriction are well known, how the increases in pressure trigger vasoconstriction is unclear. The response is initiated by pressure-induced stretch of the vessel wall and thus is dependent on mechanical signaling. The identity of the sensor detecting VSMC stretch is unknown. Previous studies have considered the role of extracellular matrix-integrin interactions, ion conduction units (channels and/or transporters), and the cytoskeleton as pressure detectors. Whether, and how, these structures fit together in VSMCs is poorly understood. However, a model of mechanotransduction in the nematode Caenorhadbditis elegans ( C. elegans) has been established that ties together extracellular matrix, ion channels, and cytoskeletal proteins into a large mechanosensing complex. In the C. elegans mechanotransducer model, a family of evolutionarily conserved proteins, referred to as the DEG/ENaC/ASIC family, form the ion-conducting pore of the mechanotransducer. Members of this protein family are expressed in VSMC where they may participate in pressure detection. This review will address how the C. elegans mechanotransducer model can be used to model pressure detection in mammalian VSMCs and provide a new perspective to pressure detection in VSMCs.

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Katanin, the microtubule-severing ATPase, is concentrated at centrosomes

Journal of Cell Science ◽

10.1242/jcs.109.3.561 ◽

1996 ◽

Vol 109 (3) ◽

pp. 561-567 ◽

Cited By ~ 18

Author(s):

F.J. McNally ◽

K. Okawa ◽

A. Iwamatsu ◽

R.D. Vale

Keyword(s):

Mitotic Spindle ◽

Dynamic Properties ◽

Mitotic Spindles ◽

Microtubule Severing ◽

Spindle Microtubules ◽

Gamma Tubulin ◽

And Function ◽

Poleward Flux

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

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Microtubule-severing activity of the AAA+ ATPase Katanin is essential for female meiotic spindle assembly

Development ◽

10.1242/dev.140830 ◽

2016 ◽

Vol 143 (19) ◽

pp. 3604-3614 ◽

Cited By ~ 16

Author(s):

Nicolas Joly ◽

Lisa Martino ◽

Emmanuelle Gigant ◽

Julien Dumont ◽

Lionel Pintard

Keyword(s):

Spindle Assembly ◽

Meiotic Spindle ◽

Aaa Atpase ◽

Microtubule Severing

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Katanin controls mitotic and meiotic spindle length

The Journal of Cell Biology ◽

10.1083/jcb.200608117 ◽

2006 ◽

Vol 175 (6) ◽

pp. 881-891 ◽

Cited By ~ 198

Author(s):

Karen McNally ◽

Anjon Audhya ◽

Karen Oegema ◽

Francis J. McNally

Keyword(s):

Chromosome Segregation ◽

Eukaryotic Cell ◽

Meiotic Spindle ◽

Mitotic Spindles ◽

Wild Type ◽

Female Meiosis ◽

C Elegans ◽

Microtubule Severing ◽

Spindle Poles ◽

Type C

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that γ-tubulin–dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.

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Evolutionarily conserved roles of Caenorhabditis elegans perilipin in lipolysis

10.7287/peerj.preprints.27467v1 ◽

2019 ◽

Author(s):

Filip Kaššák ◽

Ahmed A Chughtai ◽

Marta Kostrouchová

Keyword(s):

Neutral Lipids ◽

Physiological Role ◽

Hormone Sensitive Lipase ◽

Functional Connection ◽

Regulatory Pathways ◽

C Elegans ◽

Evolutionarily Conserved ◽

Eukaryotic Organisms

Neutral lipids and namely triacyl-glycerols (TAGs) are the prevalent excess energy storage molecules in all eukaryotic organisms. They are universally organized in active cytoplasmic organelles called lipid droplets (LDs) and their breakdown is performed and regulated in an evolutionarily conserved manner. In mammals, two distinct but inter-connected pathways are believed to mediate this catabolism: conventional cytoplasmic lipolysis with effector neutral lipases; and lipophagy, a specific kind of autophagy exploiting lysosomal acidic lipases. Central molecules in this regulation are LD-resident proteins, perilipins (PLINs). Our recent discovery of a sole PLIN orthologue in C. elegans offers a unique opportunity to study these regulatory pathways, provided that the interactive mechanisms are orthologous. To determine this, we employed classical genetics with genome editing tools and in vivo microscopy to provide three lines of evidence demonstrating the conserved role of the C. elegans perilipin. Firstly, we proved the common presence of a standard lipolytic apparatus on LDs. Next, we ascertained a functional connection between nematode PLIN-1 and the effector enzyme, hormone-sensitive lipase (HOSL-1). Finally, we identified lipophagy as a secondary lipolytic pathway, which is consistent with the mammalian model. Our data provide not only a proof of concept but also suggests interesting implications by questioning the physiological role of lipophagy in lipolysis.

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The p97-UBXN1 complex regulates aggresome formation

10.1101/2020.09.03.281766 ◽

2020 ◽

Author(s):

Sirisha Mukkavalli ◽

Jacob Aaron Klickstein ◽

Betty Ortiz ◽

Peter Juo ◽

Malavika Raman

Keyword(s):

Protein Aggregation ◽

Mammalian Cells ◽

Proteasome Inhibition ◽

Adaptor Proteins ◽

Ubiquitin Proteasome System ◽

Aaa Atpase ◽

Evolutionarily Conserved ◽

Ubiquitin Proteasome ◽

Aggresome Formation

AbstractThe recognition and disposal of misfolded proteins are essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. It is presently unclear how diverse disease-relevant aggregates are recognized and processed for degradation. The p97 AAA-ATPase in combination with a host of adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the ubiquitin-proteasome system or through autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear given the large number of pathways that rely on p97 function. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes which are membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates both aggresome formation and clearance in proteasome-inhibited cells. We identify a novel and specific role for the p97 adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to ubiquitin-positive aggresomes and UBXN1 knockout cells are unable to form a single aggresome, and instead display dispersed ubiquitin aggregates. Furthermore, loss of p97-UBXN1 results in the increase in Huntingtin polyQ aggregates both in mammalian cells as well as in a C.elegans model of Huntington’s Disease. Together our work identifies evolutionarily conserved roles for p97 and its adaptor UBXN1 in the disposal of protein aggregates.

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Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly

The Journal of Cell Biology ◽

10.1083/jcb.200812167 ◽

2009 ◽

Vol 185 (5) ◽

pp. 859-874 ◽

Cited By ~ 101

Author(s):

Ying Wai Chan ◽

Luca L. Fava ◽

Andreas Uldschmid ◽

Michael H.A. Schmitz ◽

Daniel W. Gerlich ◽

...

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Critical Role ◽

Spindle Orientation ◽

Aurora B ◽

C Elegans ◽

Spindle Rotation ◽

Mitotic Control ◽

New Protein

Mitotic spindle formation and chromosome segregation depend critically on kinetochore–microtubule (KT–MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.

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