Strehler, Emanuel E.

Myosin 10 (Myo10) is an unconventional myosin involved in a wide range of cell processes from filopodial formation and extension to spindle orientation during cell division. Among its multiple functional domains, Myo10 contains three IQ motifs that bind calmodulin and specifically calmodulin‐like protein (CLP) as light chains. We have recently shown that CLP expression up‐regulates Myo10, leading to increased Myo10‐dependent cell motility and filopodial extension (R.D. Bennett et al., J. Biol. Chem. 282, 3205–3212, 2007). CLP‐dependent Myo10 up‐regulation occurs without a concomitant increase in Myo10 mRNA. We followed Myo10 degradation in vivo and in vitro and found that CLP does not affect the decay rate of Myo10. Myo10 decayed rapidly with a half‐life of about 2.5 hours indicating that this myosin participates in signaling processes. Using an in vitro transcription/translation system we determined that CLP increased Myo10 heavy chain translation, resulting in a higher relative accumulation of Myo10 in the presence than in the absence of CLP. Taken together, our data suggest that CLP functions to increase translation of Myo10 possibly by acting as a chaperone for the emerging Myo10 heavy chain protein. Supported in part by the Mayo Graduate School and grant BCTR‐0201979 from the Susan G. Komen Breast Cancer Foundation.

Plasma Membrane Ca 2+ ATPase isoform 4b (PMCA4b) is the primary Ca 2+ efflux pump in murine sperm where it regulates motility. In Pmca4 null sperm hyperactivated motility is lost, resulting in infertility. We have shown that PMCA4b interacts with CASK in regulating Ca 2+ homeostasis and motility. Recently, it has been shown that PMCA4a and 4b are expressed in all bovine epididymal (EPL) regions, with increasing amounts of 4a distally and only 4b in testis. These findings suggest that PMCA4a and b are acquired by sperm during EPL maturation. Here we show, via RT‐PCR, that murine Pmca4b mRNA is expressed uniformly in testis and epididymis (EP) and 4a differentially in the EP. Western confirmed the RT‐PCR results showing that PMCA4a is most abundant in testis and cauda EP. Immunoprecipitation using pan‐PMCA4 antibody showed 4b and 4a in caput and corpus EP and 4a in cauda, seminal vesicle (SV), and vas deferens (VD). Western revealed an ~4 fold increase of 4a in caudal, compared to, caput sperm and confirmed its presence in the VD and SV. Importantly, PMCA4a was seen in sperm‐free EPL luminal fluid, suggesting that it is secreted, and acquired by sperm via epididymosomes. Since PMCA4a and 4b have different kinetic properties for Ca 2+ transport, our study suggests for the first time that murine sperm possess PMCA4a which has a higher Ca 2+ turnover, better facilitating capacitation events in the female tract. Funding: NIH‐RO3 HD061637.

Plasma Membrane Ca2+‐ATPase isoform 4 (PMCA4) is the primary Ca2+ efflux pump in murine sperm where it regulates motility. In Pmca4 null sperm hyperactivated motility is lost, resulting in infertility. We have shown that PMCA4b – CASK interaction in sperm is key in regulating Ca2+ homeostasis. However, recent work showed that in bulls the PMCA4a splice variant (not in testis) is epididymally‐expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT‐PCR, that both splice variants of Pmca4 mRNA are expressed in the murine testis and along the epididymis. Immunohistochemistry localized PMCA4a in the epididymal epithelium and Western analysis showed, for the first time, that PMCA4a is secreted in the epididymal luminal fluid (ELF). Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5‐fold compared to caput sperm (detected by Western) and~2‐fold and ~2.75‐fold after incubation in ELF and epididymosomes, respectively, revealing in vitro uptake. Using pan‐PMCA4 antibodies, CO‐IP assays revealed that both variants associate with CASK, indicating possible function as a complex. Our study suggests a mechanism for combining the different functional attributes of both variants, leading to greater efficiency of the pump in maintenance of Ca2+ homeostasis, which is crucial for sperm motility and male fertility.

Calcium pumps of the plasma membrane (also known as plasma membrane Ca2+-ATPases or PMCAs) are responsible for the expulsion of Ca2+from the cytosol of all eukaryotic cells. Together with Na+/Ca2+exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca2+concentration. Like the Ca2+pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca2+from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca2+pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca2+signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA “knockout” mice further support the concept of specific, nonredundant roles for each Ca2+pump isoform in cellular Ca2+regulation.

Plasma membrane Ca2+ATPases (PMCAs) are ubiquitous in Ca2+-transporting organs, including the kidney. Using RT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na+/Ca2+exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring45Ca2+flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca2+transport was provided by Na+/Ca2+exchanger and one-third by PMCAs, as determined in Na+-free media and using various PMCA inhibitors (La3+, vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca2+efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca2+transport to ∼140%, whereas antisense treatment reduced Ca2+flux ∼45% compared with controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca2+reabsorption in the distal kidney.

Plasma membrane calcium pumps differentially affect the pattern of calcium signals.

Oral cancer is often diagnosed only at advanced stages due to a lack of reliable disease markers. The purpose of this study was to determine if the epithelial-specific human calmodulin-like protein (CALML3) could be used as marker for the various phases of oral tumor progression. Immunohistochemical analysis using an affinity-purified CALML3 antibody was performed on biopsy-confirmed oral tissue samples representing these phases. A total of 90 tissue specimens were derived from 52 patients. Each specimen was analyzed in the superficial and basal mucosal cell layers for overall staining and staining of cellular subcompartments. CALML3 was strongly expressed in benign oral mucosal cells with downregulation of expression as squamous cells progress to invasive carcinoma. Based on the Cochran-Armitage test for trend, expression in the nucleus and at the cytoplasmic membrane significantly decreased with increasing disease severity. Chi-square test showed that benign tissue specimens had significantly more expression compared to dysplasia/CIS and invasive specimens. Dysplasia/CIS tissue had significantly more expression than invasive tissue. We conclude that CALML3 is expressed in benign oral mucosal cells with a statistically significant trend in downregulation as tumorigenesis occurs. CALML3 may thus be a sensitive new marker for oral cancer screening.