Acquisition Of Motility Research Articles

TMEM16A inhibition impedes capacitation and acquisition of hyperactivated motility in guinea pig sperm

Ca2+ -activated Cl- channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl- in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of ∼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca2+ ]i, and increasing [Cl- ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes.

An epididymis-specific secretory protein Clpsl2 critically regulates sperm motility, acrosomal integrity, and male fertility.

The epididymis performs an important role in the maturation of spermatozoa including their acquisition of progressive motility and fertilizing ability. However, the molecular mechanisms that govern these maturational events are still poorly defined. Here we report that Clpsl2, a novel colipase homology, is exclusively expressed in the caupt epididymis and conserved in mammalian. Clpsl2 was secreted into the lumen and covered the acrosome region and principal piece of spermatozoa tail. And during epididymal transit, the binding rate between Clpsl2 protein and the spermatozoa gradually decreased. Though Clpsl2 had the highest identifies with pancreatic colipase (Clps), Clpsl2 lacked those conserved amino acids in pancreatic Clps that interacting with lipase, correspondingly, the recombinant Clpsl2 protein did not possess the Clps function such as promoting the hydrolysis of lipase to its substrate glycerine trioleate. However, sequence analysis showed that Clpsl2 has the potency to bind with lipid. Knockdown expression of Clpsl2 by lentivirus-mediated RNAi in vivo caused an attenuation of spermatozoa motility, a suppressed acrosomal reaction, a decrease of cauda spermatozoa number, and subfertility. This study identified a novel and conserved molecule, Clpsl2, was specifically expressed in epididymis and involved in the regulation of spermatozoa motility, acrosomal integrity, and male fertility.

TrkC promotes colorectal cancer growth and metastasis.

The current work reveals that TrkC receptor is crucial to many aspects of tumorigenicity and metastasis of cancer. However, with only a few exceptions, such as colorectal cancer (CRC), where suppressing tumorigenic and metastatic ability via expression of TrkC as tumor suppressor have been proposed. These diverse lines of evidence led us to investigate whether TrkC is involved in CRC progression. By using mouse models and molecular biology analyses, we demonstrate that TrkC acts as an activator in tumorigenicity and metastasis of colorectal cancer. In this study, TrkC was frequently overexpressed in CRC cells, patients’ tumor samples and an azoxymethane/dextran sulphate sodium-induced mouse model of colitis-associated CRCs. TrkC expression was associated with a high-grade CRC phenotype, leading to significantly poorer survival. Also, TrkC expression promoted the acquisition of motility and invasiveness in CRC. Moreover, TrkC increased the ability to form tumor spheroids, a property associated with cancer stem cells. Importantly, knockdown of TrkC in malignant mouse or human CRC cells inhibited tumor growth and metastasis in a mouse xenograft model. Furthermore, TrkC enhanced metastatic potential and induced proliferation by aberrant gain of AKT activation and suppression of transforming growth factor (TGF)-β signalling. Interestingly, TrkC not only modulated the actions of TGF-β type II receptor, but also attenuated expression of this receptor. These findings reveal an unexpected physiological role of TrkC in the pathogenesis of CRC. Therefore, TrkC is a potential target for designing effective therapeutic strategies for CRC development.

Abstract B26: Long interspersed nuclear element-1 regulates malignant transformation of lung bronchial epithelial cells through epithelial-to-mesenchymal transition

Abstract Lung cancer has the highest cancer-related mortality in the United States. Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer. More than 65% of NSCLC patients show cancer progression presenting with locally advanced or metastatic disease. There is an imminent need to identify novel molecular targets that can help improve the precision of current therapies. Long interspersed nuclear element-1 (L1) is an abundant genetic element that mobilizes via retrotransposition using L1-encoded ORF1p and ORF2p proteins. L1 is reactivated by the carcinogen benzo(a)pyrene (BaP) which is a polycyclic aromatic hydrocarbon (PAH) present in tobacco smoke and environment pollutant. L1 reactivation is highly mutagenic by insertion into different locations in the genome. Recent studies have implicated L1 in the onset and progression of lung cancer, but the molecular bases of this response remain largely unknown. Moreover, the genome of lung cancer is one of the most frequently affected by L1 insertions, with reports showing that 50% of NSCLC have L1 ORF1p expression across a panel of different human lung neoplasms. L1 proteins expression correlates with downregulation of differentiation genes and appearance of undifferentiated phenotypes. Epithelial-to-mesenchymal transition (EMT) is a process that promotes undifferentiated phenotypes, loss of cell–cell adhesion and acquisition of cell motility, promoting invasiveness, metastasis and apoptotic resistance. To assess whether L1 induces EMT phenotypes to promote malignant transformation and cancer progression, non-malignant human bronchial epithelial BEAS-2B cells were stably transfected with vectors that constitutively expressed wildtype L1, a mutant counterpart that lacked reverse trascriptase activity and thus was unable to retrotranpose (mutant L1), or empty vector as control. Immunoblotting showed that cells expressing L1 and mutant L1 proteins exhibited increased expression of the mesenchymal markers, N-Cadherin and Snail, coupled with decreased expression of the epithelial marker ZO-1 compared to control cells. The expression of E-Cadherin or Vimentin was unaffected by ectopic expression of L1. Interestingly, Claudin-1 was selectively induced in cells expressing wildtype L1, but not cells expressing the mutant L1 or empty vector. The changes in ZO-1 and N-Cadherin expression, but not Claudin-1, were reversible upon genetic knockdown of L1. Importantly, ectopic expression of wildtype and mutant L1 in BEAS-2B cells enhanced tyrosine kinase inhibitor chemoresistance and induces a tumorigenicity in vivo. We conclude that L1 may contribute to lung carcinogenesis and progression by promoting cell transformation to induce tumorigenecity and changing cell plasticity to induce EMT-like phenotype via retrotransposition-dependent and -independent mechanisms in non-malignant bronchial epithelial. Citation Format: Ivan O. Aispuro, Elsa M. Reyes-Reyes, Minerva SantaCruz, Kenneth S. Ramos. Long interspersed nuclear element-1 regulates malignant transformation of lung bronchial epithelial cells through epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B26.

PARP3 controls TGFβ and ROS driven epithelial-to-mesenchymal transition and stemness by stimulating a TG2-Snail-E-cadherin axis.

Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFβ and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFβ-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFβ-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFβ-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44high/CD24low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFβ-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

PIPKIγ and talin couple phosphoinositide and adhesion signaling to control the epithelial to mesenchymal transition.

Epithelial cells acquire migratory/invasive and stemness traits upon conversion to the mesenchymal phenotype. The expression of E-cadherin is a key to this transition; yet precise understanding of the pathways involved in integrating E-cadherin loss to the gain of mesenchymal traits remains poorly understood. Here, we show that phosphoinositide-generating enzyme, PIPKIγ, expression is upregulated upon epithelial-mesenchymal transition (EMT) and together with the cytoskeletal protein talin assemble into a signaling complex upon E-cadherin loss. PIPKIγ and talin together control the adhesion and phosphoinositide signaling that regulates conversion to the mesenchymal phenotypes. PIPKIγ and talin regulate the stability of E-cadherin transcriptional repressors, snail and slug, induced by transforming growth factor-β1 or extracellular matrix protein. Loss of PIPKIγ or talin or their interaction impaired EMT and the acquisition of cell motility and stemness. This demonstrates a mechanism where a phosphoinositide-generating enzyme PIPKIγ couples with a cytoskeletal protein talin to control the acquisition of mesenchymal phenotypes.

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.

3 ROLE OF β-DEFENSIN 126 IN PROMOTING SPERM MOTILITY IN CATTLE

As immature sperm migrate through the epididymis, they are bathed in region-specific epididymal fluid, which leads to a sequential addition, deletion, and modification of their surface proteins. These changes ultimately result in the acquisition of motility and fertilising abilities. Among the hundreds of proteins secreted by the epididymis, several β-defensins have been identified and correlated with male fertility in multiple species. In cattle, β-defensin 126 (BD126) is exclusively detected in the reproductive tract of pubertal males, with preferential mRNA expression in the epididymis. Both the macaque and human orthologs have been shown to play a role in the ability of sperm to migrate through cervical mucus. The aim of this study was to examine the role of bovine BD126 in sperm function. Western blot revealed that the peptide is uniquely present in both the cauda epididymis sperm and fluid and is absent from sperm recovered from other proximal epididymal regions, or the ejaculate of vasectomised animals. Confocal analysis showed immunofluorescent labelling of BD126 specific to the tail and acrosomal region in cauda sperm only, suggesting a role in motility. We hypothesised that addition of cauda epididymal fluid (CEF) or recombinant BD126 (rBD126) to immature corpus sperm would improve ability to penetrate cervical mucus. Testes from adult bulls were collected at an abattoir, and sperm from the corpus and cauda epididymis, as well as CEF, were recovered. Corpus sperm were incubated for 1 h with CEF in the absence or presence of BD126 antibody, or with different rBD126 concentrations (30 or 60 μg mL–1); untreated corpus and cauda sperm were used as controls. A higher number of cauda than corpus sperm migrated through cervical mucus from oestrus cows (P < 0.001), and addition of CEF increased the number of corpus sperm migrating through this matrix (P < 0.05). The presence of the BD126 antibody in CEF failed to abrogate this effect. Western blot analysis of the sperm samples revealed the antibody was not successful in blocking BD126 from binding onto the sperm surface, which would explain the lack of differences observed. Furthermore, the addition of rBD126 did not increase corpus sperm migration through mucus. In conclusion, we have characterised the expression of bovine BD126 protein in the bovine testis and epididymis. Incubation of sperm from the corpus with CEF from the cauda resulted in enhanced sperm migration through cervical mucus. However, incubation of sperm with rBD126 in the absence of other factors and proteins from the CEF failed to produce the same effect. These results suggest that the role of BD126 in cattle is different from that observed in primates. We are currently investigating other roles of BD126 and related β-defensins in mediating bovine sperm function. This work was supported by a grant from the Irish Department of Agriculture, Food and The Marine under the Research Stimulus Programme (Grant No. 11S 104).

Lessons learned in andrology: looking at the spermatozoa in context.

Lessons learned in andrology: looking at the spermatozoa in context.

Discrete Functions Of GSK3α And GSK3β Isoforms In Prostate Tumor Growth And Micrometastasis

ObjectiveRole of glycogen synthase kinase‐3 (GSK3) in cancer is controversial and the isoform specific function of GSK3 in cancer is not well defined. The present study aims to explore the discrete functions of GSK3α and GSK3β in prostate cancer growth and metastasis.
Methods and ResultsWe report that silencing the expression of GSK3α or GSK3β isoforms inhibit overall growth and proliferation of the metastatic prostate cancer cells with a predominant role of GSK3α isoform in the process. Deficiency of GSK3α, but not GSK3β inhibited colony formation by the PC3 and DU145 metastatic prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Despite the modest effect of GSK3β knockdown on the cell proliferation in vitro, knockdown of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts in vivo. ShRNA‐mediated knockdown of GSK3α and GSK3β equally inhibited the ability of PC3 cells to migrate and invade the endothelial‐barrier in vitro, and micrometastasis in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro‐apoptotic markers cleaved caspase‐3 and cleaved caspase‐9 in both PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell‐cell contacts, increased expression and membrane localization of β‐catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP‐9 indicating specific role for GSK3β in EMT, and acquisition of motility and invasive potential. 
ConclusionsThe isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.

Cilia in the choroid plexus: their roles in hydrocephalus and beyond.

Cilia are whip-like projections that are widely conserved in eukaryotes and function as a motile propeller and/or sensory platform to detect various extracellular stimuli. In vertebrates, cilia are ubiquitously found in most cells, showing structural and functional diversities depending on the cell type. In this review, we focus on the structure and function of cilia in choroid plexus epithelial cells (CPECs). CPECs form one or two dozen non-motile 9+0 cilia, which display transient acquisition of motility during development. Genetic malfunction of cilia can lead to failure of multiple organs including the brain. Especially, several groups have demonstrated that the defects in CPEC cilia cause the communicating form of hydrocephalus. In order to elucidate the molecular mechanisms underlying the hydrocephalus, we have previously demonstrated that the cilia possess an NPFF receptor for autocrine signaling to regulate transepithelial fluid transport. In this perspective, we also discuss the potential involvement of cilia in the other aspects of choroid plexus functions, such as the regulation of brain development and neuroinflammation.

Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis.

Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.

270 PROGRESSIVE INCORPORATION OF CENEXIN IS RELATED TO SPERM MATURATION DURING EPIDIDYMAL TRANSIT IN THE DOMESTIC CAT

The sperm centrosome is an essential organelle playing a key role just after penetration into the oocyte. It serves to organise the sperm aster, which is required for syngamy and formation of the first mitotic spindle. It is also associated with acquisition of motility during epididymal transit. Previously, we demonstrated that testicular spermatozoa exhibit reduced developmental potential after oocyte injection due to the presence of an immature centrosome [Comizzoli et al. 2006 Biol. Reprod. 75, 252–260]. Centrosome and flagellum maturation naturally occur during epididymal transit where secreted proteins impart changes on the sperm to acquire its functional properties. The objective of this study was to better understand centrosome and flagellum maturation and identify key proteins that could be used to artificially mature testicular spermatozoa. Specifically, we focused our effort on cenexin, a protein that has been reported to aid in maturation of the flagellum and somatic cell centrosome. Epididymides were dissected from adult cat testes (>1 yr old). Spermatozoa were then extracted from the different regions (caput, corpus, cauda, and vas deferens) by slicing with a scalpel blade in phosphate buffered saline at 37°C and processed separately. Control samples were also collected from the rete testis. After recording sperm motility and forward progressive movement (FPM, from 0 = immotile to 5 = fast and straight), cells were fixed in 4% paraformaldehyde and immunostained with anti-cenexin antibodies labelled with a fluorescent probe. The proportion of cells with cenexin at the location of the centrosome and the intensity of immunofluorescence were quantified (n = 8 and 4 testes, respectively). The same methods were followed for detection of cenexin in the tail portion (n = 4 testes). Statistical analyses were conducted using repeated-measures and treatments were further compared using either a protected Tukey's or F-test for orthogonal contrasts. The proportion of sperm with cenexin localised at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis and highest proportion in the cauda (45 v. 81%, T28 = 4.65, P < 0.0001). Among the labelled sperm, the intensity of immunofluorescence also significantly increased from the testis to vas deferens (4.33 v. 8.57 mean grey value; T12 = 3.29, P < 0.0065). Both motility and FPM increased from the testis to cauda segment (0 v. 93%, F4,15 = 13.53, P < 0.0001 and 0 v. 3.8 FPM, F4,15 = 26.67, P < 0.0001); however, the proportion with cenexin in the tail (range, 20 to 36%) as well as the labelling intensity (range, 3.14 to 5.26 mean grey value) did not change (P > 0.05) along the tract. These results clearly indicate that cenexin may be associated with centrosome but not flagellum maturation. Epididymal epithelial cells and luminal fluid from each segment are being examined to better understand the source of cenexin secretion and its incorporation into spermatozoa. Results from these studies will aid in further understanding the physiology of sperm maturation during epididymal transit and increase male fertility preservation options.

Intravital imaging of SRF and Notch signalling identifies a key role for EZH2 in invasive melanoma cells.

The acquisition of cell motility is an early step in melanoma metastasis. Here we use intravital imaging of signalling reporter cell-lines combined with genome-wide transcriptional analysis to define signalling pathways and genes associated with melanoma metastasis. Intravital imaging revealed heterogeneous cell behaviour in vivo: less than 10% of cells were motile and both singly moving cells and streams of cells were observed. Motile melanoma cells had increased Notch- and SRF-dependent transcription. Subsequent genome-wide analysis identified an overlapping set of genes associated with high Notch and SRF activity. We identified EZH2, a histone methyltransferase in the Polycomb Repressor Complex 2, as a regulator of these genes. Heterogeneity of EZH2 levels is observed in melanoma models and co-ordinated up-regulation of genes positively regulated by EZH2 is associated with melanoma metastasis. EZH2 was also identified as regulating the amelanotic phenotype of motile cells in vivo by suppressing expression of the P-glycoprotein Oca2. Analysis of patient samples confirmed an inverse relationship between EZH2 levels and pigment. EZH2 targeting with siRNA and chemical inhibition reduced invasion in mouse and human melanoma cell lines. The EZH2 regulated SRF target genes KIF2C and KIF22 are required for melanoma cell invasion and important for lung colonisation. We propose that heterogeneity in EZH2 levels leads to heterogeneous expression of a cohort of genes associated with motile behaviour including KIF2C and KIF22. EZH2 dependent increased expression of these genes promotes melanoma cell motility and early steps in metastasis.

Temporal control over the initiation of cell motility by a regulator of G-protein signaling.

The control over the acquisition of cell motility is central for a variety of biological processes in development, homeostasis, and disease. An attractive in vivo model for investigating the regulation of migration initiation is that of primordial germ cells (PGCs) in zebrafish embryos. In this study, we show that, following PGC specification, the cells can polarize but do not migrate before the time chemokine-encoded directional cues are established. We found that the regulator of G-protein signaling 14a protein, whose RNA is a newly identified germ plasm component, regulates the temporal relations between the appearance of the guidance molecules and the acquisition of cellular motility by regulating E-cadherin levels.

SLC6 family transporter SNF-10 is required for protease-mediated activation of sperm motility in C. elegans

Motility of sperm is crucial for their directed migration to the egg. The acquisition and modulation of motility are regulated to ensure that sperm move when and where needed, thereby promoting reproductive success. One specific example of this phenomenon occurs during differentiation of the ameboid sperm of Caenorhabditis elegans as they activate from a round spermatid to a mature, crawling spermatozoon. Sperm activation is regulated by redundant pathways to occur at a specific time and place for each sex. Here, we report the identification of the solute carrier 6 (SLC6) transporter protein SNF-10 as a key regulator of C. elegans sperm activation in response to male protease activation signals. We find that SNF-10 is present in sperm and is required for activation by the male but not by the hermaphrodite. Loss of both snf-10 and a hermaphrodite activation factor render sperm completely insensitive to activation. Using in vitro assays, we find that snf-10 mutant sperm show a specific deficit in response to protease treatment but not to other activators. Prior to activation, SNF-10 is present in the plasma membrane, where it represents a strong candidate to receive signals that lead to subcellular morphogenesis. After activation, it shows polarized localization to the cell body region that is dependent on membrane fusions mediated by the dysferlin FER-1. Our discovery of snf-10 offers insight into the mechanisms differentially employed by the two sexes to accomplish the common goal of producing functional sperm, as well as how the physiology of nematode sperm may be regulated to control motility as it is in mammals.

Changes of murine sperm phospholipid composition during epididymal maturation determined by MALDI-TOF mass spectrometry

After leaving the testis, spermatozoa undergo several important steps of biochemical maturation during the passage through the epididymis, increasing their motility and fertilizing ability. These changes comprise (among others) the modification of the phospholipid composition of the sperm membrane. This process is thought to be important for the achievement of motility and fertilizing capacity. The lipids of the sperm membrane are characterized by a significant content of unsaturated fatty acyl residues, resulting in a high sensitivity against oxidative stress. This is evidenced by the appearance of lysolipids, for example, lysophosphatidylcholine, which acts like a detergent and is normally present in only very small amounts in biological membranes. The epididymis represents a tubular system comprising three main parts (caput, corpus, and cauda), through which the spermatozoa are consecutively transported undergoing distinct maturation stages. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we established three striking differences in the lipid composition of murine spermatozoa from the different epididymal regions: in comparison to the caput sperm, sperm from the cauda are characterized by (1) a higher degree of unsaturation (PC 18:0/22:5 and 18:0/22:6 vs. 18:0/20:4 and 18:0/18:1), (2) an enhanced plasmalogen content, and (3) an enhanced content of lysolipids. These changes are likely to be of physiological relevance and potentially useful as diagnostic markers of sperm maturation and acquisition of motility.

ROS-induced epithelial-mesenchymal transition in mammary epithelial cells is mediated by NF-kB-dependent activation of Snail.

Epithelial-mesenchymal transition (EMT) is characterized by loss of cell-cell junctions, polarity and epithelial markers, and in turn, acquisition of mesenchymal features and motility. Changes associated with this developmental process have been extensively implicated in breast cancer progression and metastasis. Matrix metalloproteinases (MMPs) have been identified as specific inducers of EMT in mammary epithelial cells. MMP-3 induces EMT associated with malignant transformation via a pathway dependent upon production of reactive oxygen species (ROS). While the process by which exposure to MMP-3 leads to induction of ROS has been extensively studied, exactly how the MMP-3-induced ROS stimulate EMT remains unknown. Here, we used profiling methods to identify MMP-3-induced transcriptional alterations in mouse mammary epithelial cells, finding common overlap with changes mediated by nuclear factor-κB (NF-κB) and found in advanced breast cancer. In cultured cells, we found that Snail, an ROS-dependent key mediator of MMP-3-induced changes, is regulated by NF-κB in response to MMP-3. More specifically, we found MMP-3 to cause binding of p65 and cRel NF-κB subunits to the Snail promoter, leading to its transcription. Our results identify a specific pathway by which MMPs induce EMT and malignant characteristics, and provide insight into potential therapeutic approaches to target MMP-associated breast cancers.

Stromal exosomes allow cancer cell autoactivation

Exosomes are small vesicles derived from endosomes and carrying several constituants of the cell; if captured by other neighbour cell types, they can trigger novel functions in these cells. We illustrate here (through recently published results) how exosomes released by activated tumor-associated fibroblasts are able to induce in cancer cells a signalling pathway key to the acquisition of motility and hence metastatic property.

Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion

BackgroundUnraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process.MethodsTo investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ.ResultsDGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro. DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated their invasiveness.ConclusionElevated DGKζ expression contributes to increased Rho GTPase activation and the enhanced motility of metastatic cancer cells. These findings warrant further investigation of the clinical relevance of DGKζ upregulation in colon and other cancers. Interfering with DGKζ function could provide a means of inhibiting invasion and metastasis.