Fernando, Lourds et al. (2020) MicroPubl Biol

Nguyen, Victoria, Allen, Anna, Hansen, Tyler, Golden, Andy, Fernando, Lourds

[MicroPubl Biol, 2020]

The 26S proteasome is one of the major proteolytic machineries in cells and is composed of nearly 33 different subunits (Budenholzer et al., 2017; Chen et al., 2008; Papaevgeniou and Chondrogianni, 2014). The subunits are arranged as one or two 19S regulatory particle(s) capping a cylindrical catalytic 20S core particle (Budenholzer et al., 2017). Each subunit is highly conserved from yeast to mammals, and proper function of the proteasome is crucial for survival of organisms (Papaevgeniou and Chondrogianni, 2014). Spatiotemporal expression of specific subunits and their roles in regulating the proteasome activity is still not clearly understood. RPN-12 is a subunit of the 19S regulatory particle (RP) of the 26S proteasome in Caenorhabditis elegans (Boehringer et al., 2012; Takahashi et al., 2002). In C. elegans, loss or down regulation of a single proteasome subunit causes embryonic lethality, with the exception of three of the 19S RP subunits: RPN-10, RPN-12, and DSS-1 (Keith et al., 2016; Pispa et al., 2008; Shimada et al., 2006; Takahashi et al., 2002). While the roles of RPN-10 and DSS-1 were previously studied, more detailed investigation into the function of RPN-12 has not been performed. A heterozygous mutant containing a 952bp deletion of the coding region of rpn-12 was generated [rpn-12(av93)] using CRISPR/Cas9 genome editing technology. The heterozygous rpn-12 null mutant was then homozygosed and determined to be homozygous viable with apparent fertility issues. Only 36% of the rpn-12(av93) hermaphrodites produced progeny due to sperm production defects (this phenotype will be published elsewhere). Since efficient proteasome activity is important for the longevity of most organisms, we wanted to investigate whether rpn-12(av93) mutants may have an altered lifespan compared to wild type animals (Saez and Vilchez, 2014; Vilchez et al., 2012). To determine whether the lifespan of rpn-12(av93) mutant animals was affected, lifespan assays were conducted at 20C and 25C. The mean lifespan of rpn-12(av93) hermaphrodites (15.94 days) was not significantly different from wild type hermaphrodites (15.54 days) at 20C. Interestingly, at 25C the mean lifespan of rpn-12(av93) hermaphrodites (15.59 days) was increased compared to wild type animals (12.80 days). However, the maximum lifespan of both rpn-12(av93) and wild type hermaphrodites was not significantly different at 25C. The lifespan analysis at 20C was performed three times (cumulative wild type n=240 and mutant n=192) and at 25C was performed twice (cumulative wild type n=185 and mutant n=135). Our data suggests that RPN-12 is not essential for viability and lifespan of C. elegans under normal conditions (20C), but that absence of RPN-12 can result in a significant increase in mean lifespan under heat stress (25C).

Eto K et al. (1999) J Biol Chem "Ca(2+)/Calmodulin-dependent protein kinase cascade in Caenorhabditis elegans. Implication in transcriptional activation."

Kimura Y, Eto K, Tokumitsu H, Arai K, Takahashi N, Muramatsu M, Masuho Y

[J Biol Chem, 1999]

We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

Park, Aileen et al. (2020) MicroPubl Biol

Qiu, Zhongqiang, Cross, Margaret, Brice, Sophia, Whiteley, Lauren, Lee, Myeongwoo, Baur, Olivia, Park, Aileen, Lewis, Kendall

[MicroPubl Biol, 2020]

The him-4 gene in Caenorhabditis elegans is involved in important developmental processes in the organism including basement membrane attachment, anchor cell invasion, defective cytokinesis, and gonad positioning along the basement membrane (Morrissey et al., 2014). The human ortholog to him-4, HMCN 1 (hemicentin-1), is implicated in age related macular degeneration 1 (Thompson et al., 2007). HIM-4/HMCN1 is a highly conserved extracellular matrix structural component and contains six RGD (Arg-Gly-Asp) cell-binding motifs within its 48 immunoglobin (Ig)-like repeats, while HMCN1 contains only one RGD motif (Vogel and Hedgecock, 2001). Two novel mutant alleles of C. elegans him-4 were generated using CRISPR-Cas9 gene editing in this study. In one mutant allele, three of the aforementioned six RGD motifs were mutated to RGE (Arg-Gly-Glu) (Takahashi et al., 2007). Four of the six RGD motifs were altered to RGE in the second mutant line. Neither of these mutant lines exhibited outstanding abnormal phenotypes when screened for behavioral or morphological abnormalities. We examined the mutants for Him (high incidence in male) phenotype, kq8207 (0% male, n=888) and kq8297 (0.1% male, n=963). However, the examined animals failed to show an outstanding number of males compared to N2 (0%, n=582). Mutants were also examined for behavioral abnormalities in a thrashing assay. A Mann-Whitney U Test revealed possible differences between kq8207 mutants and N2 worms (p=0.037), and revealed no significant difference between kq8297 mutants and N2 worms (p=0.447). 53 triple RGD mutants (average 19.85 thrashes), 51 RGD quadruple mutants (average 21 thrashes), and 51 N2 worms (average 22.16 thrashes) were used in this assay. Morrissey et al. demonstrated that PAT-3/INA-1integrin is essential for assembly of HIM-4/hemicentin puncta during anchor cell invasion (Morrissey et al., 2014). However, integrin and HIM-4 failed to colocalize in other tissues (Vogel and Hedgecock 2001; Morrissey et al., 2014). Although anchor cell invasion was not examined in this study, these him-4 RGD mutants hold potential for further research pertaining to the functions of cell to extracellular matrix binding domains in C. elegans.

Wilsey, Rachel et al. MicroPubl Biol

Jaffery, Roshni, Wahl, Jacob, Lee, Myeongwoo, Brau, Avery, Kenney, Krysta, Wilsey, Rachel, Qiu, Zhongqiang, Hodge, Sabrina

[MicroPubl Biol, 2020]

The unc-52 gene in Caenorhabditis elegans encodes for the protein UNC-52 and is a homolog of the mammalian gene Heparan Sulfate Proteoglycan 2 (HSPG2), which encodes for the protein perlecan (Rogalski et al., 1993; Mullen et al., 1999). HSPG2 is implicated in the human diseases Schwartz-Jampel Syndrome type 1 and Dyssegmental Dysplasia, Silverman-Handmaker type (Arikawa-Hirasawa et al., 2001; Stum et al., 2006). The mutated unc-52 gene expresses the phenotype for uncoordinated movement (Unc), which involves progressive paralysis and retarded sarcomere construction (Martinez et al., 2018). The UNC-52 protein is localized in the striated muscle dense bodies and the basement membrane, where it plays an important role in developmental processes such as cell adhesion, cell migration, and signal transduction (Kihira et al., 2012). Within the unc-52 gene, an RGD (Arg- Gly- Asp) sequence is located at amino acid locations 746, 747, and 748 in exon 7. Exon 7, containing RGD748, is included in all three major isoforms, short (S), medium (M), and long (L) (Mullen et al., 1999). The RGD sequence is part of the Laminin IV type A domain and primarily functions as a cell attachment and adhesion site for integrins (Rogalski et al., 1993; Mullen et al., 1999). In this study, two separate mutations were performed on this unc-52 RGD sequence using CRISPR-Cas9 technology. The unc-52(kq748) mutation replaced the aspartic acid (D) located at the 748 amino acid position with a glutamic acid (E) (Takahashi et al., 2007). The unc-52(kq745) mutation removed the RGD sequence by deleting amino acids 746, 747, and 748. Previous studies have shown that unc-52 gene mutations cause the disorganized distribution of pat-3 integrin, which is a receptor for extracellular matrix proteins (Rogalski et al., 1995). In order to study the cellular phenotypes of the unc-52(kq748) mutation, a double mutant was created: pat-3::GFP; unc-52 (kq748). Staining showed that localization of the pat-3::GFP reporter in the double RGD mutant appeared normal, with dense bodies and M-lines alternating along the muscle filaments (Figures 1A and 1B). In order to visualize the actin filaments in the body wall muscles of N2 wild type and unc-52(kq748) mutants, staining was performed using 0.4 U/mL rhodamine-conjugated phalloidin, Thermo Fisher Scientific, Waltham, MA (Figures 1C and 1D). This unc-52(kq748) staining showed no obvious abnormalities outside of interruptions due to fixation and slicing procedures (Figure 1D). Additional assays were performed to investigate any other physical or behavioral phenotypes. The thrashing assay, in which worms were placed in drops of M9 buffer and the number of thrashes were counted for 15 seconds, showed a 27% average decrease in thrashes per second for the unc-52(kq748) mutants compared to the N2 worms, and a 33% decrease in unc-52(kq745) mutants (Figure 1E). A one-way ANOVA statistical test was performed to check the statistical significance of these results. The p-value from the ANOVA test was 3.1×10-9, which is less than the 0.05 needed to be statistically significant. Post hoc testing was then done to see which groups differed. Both the unc-52(kq748) and unc-52(kq745) groups differed significantly from the N2 wild-type group, as shown by p-values of 7.1×10-6 and 4.63×10-8 respectively. These results show that there was a statistically significant decrease in motility for both unc-52(kq748) and unc-52(kq745) mutants.