Naert K et al. (1997) International C. elegans Meeting "GETTING CLOSER TO KINESIN unc-116 FUNCTION."

Naert K, Thierry-Mieg D

[International C. elegans Meeting, 1997]

We will present the molecular characterization of five alleles of the kinesin heavy chain unc-116 (see wbg14.2, p84). Three of them, f130, rh24 (from Ed Hedgecock) and rh24sb79 (from Paul Mains) contain respectively one, two and three aminoacid changes in the motor domain, and produce normal amounts of mutant kinesin protein. Allele e2310 encodes a protein with an insertion of 8 aminoacids just before the cargo binding tail domain. f122 encodes a truncated kinesin with a complete motor and neck, but no rodII or tail. All alleles show maternal effects, indicating that kinesin may be stored in oocytes. We looked at meiosis and the first two embryonic mitoses in the mutants, using DAPI staining and immunofluorescence with anti-tubulin and anti P-granules. Somewhat surprisingly, we discovered that the unc-116 kinesin plays a role in mitoses with defects ranging from misplacement of nuclei to problems in the orientation, establishment and organization of the mitotic spindle. In the weak allele f130, the oocyte nucleus fails to move to the anterior pole upon fertilization; meiosis is central, the polar bodies are often not successfully ejected, hence 60% of the embryos die. Paternal rescue leads instead to dominant sterile hyperaneuploid f130/+ adults. The neomorph allele rh24 has the opposite effect of preventing pronuclear migration in 80% of the embryos. Amusingly, the combination of these two sterile alleles, f130/rh24, is fertile! Our aim is to describe accurately the functions of the kinesin heavy chain on cell morphology, membrane trafic and mitosis. We wish to propose a mechanistic explanation accounting for the genetics data including loss and gain of function effects. The four anti-kinesin antisera may help us achieve this goal.

Naert K et al. (1996) Worm Breeder's Gazette "PROGRESS ON UNC-116, WHICH ENCODES KINESIN HEAVY CHAIN"

Thierry-Mieg D, Naert K

[Worm Breeder's Gazette, 1996]

Kinesin is a microtubule-based molecular motor, driven by ATP and involved in the anterograde transport of vesicles or material. Little is know on its actual in vivo role and cargos. We have been pursuing the full genetic characterization of the four alleles of unc-116, three of which have, in addition to hypomorphic characters, specific neomorphic features, such as - maternal effect lethality in rh24, due to lack of migration of the female pronucleus in the one cell embryo (like zyg-9) ; - zygotic L1 lethality in f122 ; - and zygotic and maternal lethality in f130, linked to the generation of aneuploids with very high chromosome numbers. We are truly interested in understanding in every detail the meaning of these observations on how kinesin operates in its wild type and mutant forms, so we are identifying the molecular lesions in the mutants. Only one allele, e2310, (Patel et al, PNAS 90, 9181-85,1993) behaves as a simple and very partial loss of function. The cellular defects shared by this allele and others reflect the wild type function of kinesin, which seems ubiquitous all cell types are affected. The shape of cells, especially elongated ones (neurons, excretory canal, tail spike or male tail), is deformed, mimicking a guidance defect. The position of vesicles in the cells is often abnormal (nuclei in hyp7, or vesicles in muscle cells or oocytes), and there are severe defects in the excretory pathway leading to the accumulation of droplets between cells and basement membranes or in the pseudocoelom. The molecular defect in e2310 is an insertion of TC5 right at the boundary between rod II and tail, which should, by mental translation, lead to a kinesin devoid of its cargo holding piece. So the weakness of the allele was very puzzling. We thus performed a Northern analysis which showed the presence of two very strong bands in e2310 a transcript of about 7 kb probably corresponding to the kinesin-TC5 fusion and encoding a truncated kinesin, and another band at about the wild type size (~ 3.5 kb). Two size variants were indeed identified in the RT-PCR products corresponding to the approximately wild type band ; the longer was apparently a mixture while the shorter differs from the wild type kinesin transcript by the addition of exactly 24 bp from TC5 translation of this spliced transcript should yield a complete kinesin molecule with an added 8 aa at the rod II-tail junction, which would be consistent with the hypomorphic phenotype. Antibodies raised against peptides should allow us to verify our hypothesis. We hope to say more soon on the other alleles.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Huang CY et al. (2012) PLoS One "C. elegans EIF-3.K promotes programmed cell death through CED-3 caspase."

Tan CH, Huang CY, Wu SC, Chen JY, Wu YF, Wu YC, Chen RH, Lu PJ, Tzeng RY

[PLoS One, 2012]

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.

Wojtovich AP et al. (2011) PLoS One "SLO-2 is cytoprotective and contributes to mitochondrial potassium transport."

Sherman TA, Nehrke K, Urciuoli WR, Wojtovich AP, Nadtochiy SM, Brookes PS

[PLoS One, 2011]

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance "big" K(+) channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K(+) transport and APC. The canonical Ca(2+)-activated BK (or "maxi-K") channel SLO1 was dispensable for both mitochondrial K(+) transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K(+) channel SLO2 was required, and that SLO2-dependent mitochondrial K(+) transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K(+).

Johnson CK et al. (2020) J Neurophysiol "The Na+/K+-ATPase is needed in glia of touch receptors for responses to touch in C. elegans."

Wang Y, Bianchi L, Fernandez-Abascal J, Johnson CK, Wang L

[J Neurophysiol, 2020]

Four of the five types of mammalian mechanosensors are composed of nerve endings and accessory cells. In C. elegans we showed that glia supports the function of nose touch neurons via the activity of glial Na⁺ and K⁺channels. We show here that a third regulator of Na⁺ and K⁺, the Na⁺/K⁺-ATPase, is needed in glia of nose touch neurons for touch. Importantly, we show that the two Na⁺/K⁺-ATPase genes are needed for the function rather than structural integrity and that their ion transport activity is crucial for touch. Finally, when glial Na⁺/K⁺-ATPase genes are knocked-out, touch can be restored by activation of a third Na⁺/K⁺-ATPase. Taken together, these data show the requirement in glia of touch neurons of the function of the Na⁺/K⁺-ATPase. These data underscore the importance of the homeostasis of Na⁺ and K⁺, most likely in the space surrounding touch neurons, in touch sensation, a function that might be conserved across species.

Jospin M et al. (2002) J Physiol "Characterization of K(+) currents using an in situ patch clamp technique in body wall muscle cells from Caenorhabditis elegans."

Jospin M, Allard B, Segelat L, Mariol MC, Segalat L

[J Physiol, 2002]

The properties of K+ channels in body wall muscle cells acutely dissected from the nematode Caenorhabditis elegans were investigated at the macroscopic and unitary level using an in situ patch clamp technique. In the whole-cell configuration, depolarizations to potentials positive to -40 mV gave rise to outward currents resulting from the activation of two kinetically distinct voltage-dependent K+ currents: a fast activating and inactivating 4-aminopyridine-sensitive component and a slowly activating and maintained tetraethylammonium-sensitive component. In cell-attached patches, voltage-dependent K+ channels, with unitary conductances of 34 and 80 pS in the presence of 5 and 140 mm external K+, respectively, activated at membrane potentials positive to -40 mV. Excision revealed that these channels corresponded to Ca2+-activated K+ channels exhibiting an unusual sensitivity to internal Cl- and whose activity progressively decreased in inside-out conditions. After complete run-down of these channels, one third of inside-out patches displayed activity of another Ca2+-activated K+ channel of smaller unitary conductance (6 pS at 0 mV in the presence of 5 mm external K+). In providing a detailed description of native K+ currents in body wall muscle cells of C. elegans, this work lays the basis for further comparisons with mutants to assess the function of K+ channels in this model organism that is highly amenable to molecular and classical genetics.

Shibuya Y et al. (2022) Algorithms Mol Biol "Space-efficient representation of genomic k-mer count tables."

Kucherov G, Shibuya Y, Belazzougui D

[Algorithms Mol Biol, 2022]

MOTIVATION: k-mer counting is a common task in bioinformatic pipelines, with many dedicated tools available. Many of these tools produce in output k-mer count tables containing both k-mers and counts, easily reaching tens of GB. Furthermore, such tables do not support efficient random-access queries in general. RESULTS: In this work, we design an efficient representation of k-mer count tables supporting fast random-access queries. We propose to apply Compressed Static Functions (CSFs), with space proportional to the empirical zero-order entropy of the counts. For very skewed distributions, like those of k-mer counts in whole genomes, the only currently available implementation of CSFs does not provide a compact enough representation. By adding a Bloom filter to a CSF we obtain a Bloom-enhanced CSF (BCSF) effectively overcoming this limitation. Furthermore, by combining BCSFs with minimizer-based bucketing of k-mers, we build even smaller representations breaking the empirical entropy lower bound, for large enough k. We also extend these representations to the approximate case, gaining additional space. We experimentally validate these techniques on k-mer count tables of whole genomes (E. Coli and C. Elegans) and unassembled reads, as well as on k-mer document frequency tables for 29 E. Coli genomes. In the case of exact counts, our representation takes about a half of the space of the empirical entropy, for large enough k's.

Srivastava S et al. (2005) Mol Cell Biol "The phosphatidylinositol 3-phosphate phosphatase myotubularin- related protein 6 (MTMR6) is a negative regulator of the Ca2+-activated K+ channel KCa3.1."

Liu G, Li Z, Ko K, Skolnik EY, Coetzee WA, Srivastava S, Lin L

[Mol Cell Biol, 2005]

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3' position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P(2). MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca(2+)-activated K channel, K(Ca)3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K(Ca)3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K(Ca)3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K(Ca)3.1, indicating that MTM CC domains are sufficient to confer target specificity. K(Ca)3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K(Ca)3.1 by MTMR6 overexpression. These data, when taken together, indicate that K(Ca)3.1 is regulated by PI(3)P and that MTMR6 inhibits K(Ca)3.1 by dephosphorylating the 3' position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K(Ca)3.1. K(Ca)3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K(Ca)3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.