siRNA-Mediated MELK Knockdown Induces Accelerated Wound Healing with Increased Collagen Deposition.

Skin wounds remain a significant problem for the healthcare system, affecting the clinical outcome, patients' quality of life, and financial costs. Reduced wound healing times would improve clinical, economic, and social aspects for both patients and the healthcare system. Skin wound healing has been studied for years, but effective therapy that leads to accelerated wound healing remains to be discovered. This study aimed to evaluate the potential of MELK silencing to accelerate wound healing. A vectorless, transient knockdown of the MELK gene using siRNA was performed in a murine skin wound model. The wound size, total collagen, type 3 collagen, vessel size, vessel number, cell proliferation, cell apoptosis, number of mast cells, and immune infiltration by CD45, CD11b, CD45, and CD8a cells were evaluated. We observed that treatment with MELK siRNA leads to significantly faster wound closing associated with increased collagen deposition.

Differential Effects of Overexpression of Wild Type and Kinase-Dead MELK in Fibroblasts and Keratinocytes, Potential Implications for Skin Wound Healing and Cancer.

Maternal embryonic leucine-zipper kinase (MELK) plays a significant role in cell cycle progression, mitosis, cell migration, cell renewal, gene expression, embryogenesis, proliferation, apoptosis, and spliceosome assembly. In addition, MELK is known to be overexpressed in multiple types of cancer and is associated with cancer proliferation. Tumorigenesis shares many similarities with wound healing, in which the rate of cell proliferation is a critical factor. Therefore, this study aimed to determine the involvement of MELK in the regulation of cell division in two cell types involved in this process, namely fibroblasts and keratinocytes. We examined how temporal overexpression of wild-type and kinase-dead MELK kinase variants affect the rate of proliferation, viability, cell cycle, and phosphorylation state of other kinases involved in these processes, such as ERK1/2, AKT1, MAPK9, p38, and p53. We explored if MELK could be used as a therapeutic stimulator of accelerated wound healing via increased proliferation. We observed that aberrant expression of MELK results in abnormal proliferation, altered cell cycle distribution, and decreased viability of the cells, which challenge the utility of MELK in accelerated wound healing. Our results indicate that, at least in healthy cells, any deviation from precisely controlled MELK expression is harmful to fibroblasts and keratinocytes.

Adherens junctions are involved in polarized contractile ring formation in dividing epithelial cells of Xenopus laevis embryos.

Cells dividing in the plane of epithelial tissues proceed by polarized constriction of the actomyosin contractile ring, leading to asymmetric ingression of the plasma mem brane. Asymmetric cytokinesis results in the apical positioning of the actomyosin contractile ring and ultimately of the midbody. Studies have indicated that the contractile ring is associated with adherens junctions, whose role is to maintain epithelial tissue cohesion. However, it is yet unknown when the contractile ring becomes associated with adherens junctions in epithelial cells. Here, we examined contractile ring formation and activation in the epithelium of Xenopus embryos and explored the implication of adherens junctions in the contractile ring formation. We show that accumulation of proteins involved in contractile ring formation and activation is polarized, starting at apical cell-cell contacts at the presumptive division site and spreading within seconds towards the cell basal side. We also show that adherens junctions are involved in the kinetics of contractile ring formation. Our study reveals that the link between the adherens junctions and the contractile ring is established from the onset of cytokinesis.

Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue.

Epithelia are layers of polarised cells tightly bound to each other by adhesive contacts. Epithelia act as barriers between an organism and its external environment. Understanding how epithelia maintain their essential integrity while remaining sufficiently plastic to allow events such as cytokinesis to take place is a key biological problem. In vertebrates, the remodelling and reinforcement of adherens junctions maintains epithelial integrity during cytokinesis. The involvement of tight junctions in cell division, however, has remained unexplored. Here, we examine the role of tight junctions during cytokinesis in the epithelium of the Xenopus laevis embryo. Depletion of the tight junction-associated proteins ZO-1 and GEF-H1 leads to altered cytokinesis duration and contractile ring geometry. Using a tension biosensor, we show that cytokinesis defects originate from misregulation of tensile forces applied to adherens junctions. Our results reveal that tight junctions regulate mechanical tension applied to adherens junctions, which in turn impacts cytokinesis.This article has an associated First Person interview with the first author of the paper.

Transcriptome profiling reveals male- and female-specific gene expression pattern and novel gene candidates for the control of sex determination and gonad development in Xenopus laevis.

Xenopus laevis is an amphibian (frog) species widely used in developmental biology and genetics. To unravel the molecular machinery regulating sex differentiation of Xenopus gonads, we analyzed for the first time the transcriptome of developing amphibian gonads covering sex determination period. We applied microarray at four developmental stages: (i) NF50 (undifferentiated gonad during sex determination), (ii) NF53 (the onset of sexual differentiation of the gonads), (iii) NF56 (sexual differentiation of the gonads), and (iv) NF62 (developmental progression of differentiated gonads). Our analysis showed that during the NF50, the genetic female (ZW) gonads expressed more sex-specific genes than genetic male (ZZ) gonads, which suggests that a robust genetic program is realized during female sex determination in Xenopus. However, a contrasting expression pattern was observed at later stages (NF56 and NF62), when the ZW gonads expressed less sex-specific genes than ZZ gonads, i.e., more genes may be involved in further development of the male gonads (ZZ). We identified sexual dimorphism in the expression of several functional groups of genes, including signaling factors, proteases, protease inhibitors, transcription factors, extracellular matrix components, extracellular matrix enzymes, cell adhesion molecules, and epithelium-specific intermediate filaments. In addition, our analysis detected a sexually dimorphic expression of many uncharacterized genes of unknown function, which should be studied further to reveal their identity and if/how they regulate gonad development, sex determination, and sexual differentiation. Comparison between genes sex-specifically expressed in developing gonads of Xenopus and available transcriptome data from zebrafish, two reptile species, chicken, and mouse revealed significant differences in the genetic control of sex determination and gonad development. This shows that the genetic control of gonad development is evolutionarily malleable.

Tight junction-associated protein GEF-H1 in the neighbours of dividing epithelial cells is essential for adaptation of cell-cell membrane during cytokinesis.

Animal cells divide by a process called cytokinesis which relies on the constriction of a contractile actomyosin ring leading to the production of two daughter cells. Cytokinesis is an intrinsic property of cells which occurs even for artificially isolated cells. During division, isolated cells undergo dramatic changes in shape such as rounding and membrane deformation as the division furrow ingresses. However, cells are often embedded in tissues and thus are surrounded by neighbouring cells. How these neighbours might influence, or might themselves be influenced by, the shape changes of cytokinesis is poorly understood in vertebrates. Here, we show that during cytokinesis of epithelial cells in the Xenopus embryo, lateral cell-cell contacts remain almost perpendicular to the epithelial plane. Depletion of the tight junction-associated protein GEF-H1 leads to a transient and stereotyped deformation of cell-cell contacts. Although, this deformation occurs only during cytokinesis, we show that it originates from immediate neighbours of the dividing cell. Moreover, we show that exocyst and recycling endosome regulation by GEF-H1 are involved in adaptation of cell-cell contacts to deformation. Our results highlight the crucial role of tight junctions and GEF-H1 in cell-cell contact adaptation when cells are exposed to a mechanical stress such as cytokinesis.

Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix.

Information on the mechanisms orchestrating sexual differentiation of the bipotential gonads into testes or ovaries in amphibians is limited. The aim of this study was to investigate the development of Xenopus laevis gonad, to identify the earliest signs of sexual differentiation, and to describe mechanisms driving these processes. We used light and electron microscopy, immunofluorescence and cell tracing. In order to identify the earliest signs of sexual differentiation the sex of each tadpole was determined using genotyping with the sex markers. Our analysis revealed a series of events participating in the gonadal development, including cell proliferation, migration, cell adhesion, stroma penetration, and basal lamina formation. We found that during the period of sexual differentiation the sites of intensive cell proliferation and migration differ between male and female gonads. In the differentiating ovaries the germ cells remain associated with the gonadal surface epithelium (cortex) and a sterile medulla forms in the ovarian center, whereas in the differentiating testes the germ cells detach from the surface epithelium, disperse, and the cortex and medulla fuse. Cell junctions that are more abundant in the ovarian cortex possibly can favor the persistence of germ cells in the cortex. Also the stroma penetrates the female and male gonads differently. These finding indicate that the crosstalk between the stroma and the coelomic epithelium-derived cells is crucial for development of male and female gonad.

Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma.

BACKGROUND: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in cell cycle, differentiation, proliferation, and apoptosis. These multiple features are consistent with it being a potential anticancer target. Nevertheless, the MELK pathway in tumorigenesis is not yet completely understood. This study aims to identify proteins associated with MELK pathway in astrocytomas. To this end, proteomic data of the human glioma cell line U87MG transfected with siRNA for MELK were compared with non-target transfected control cells and compared with oligonucleotide microarray data. RESULTS: In both assays, we identified stathmin/oncoprotein 18 (STMN1), involved in cell cycle. STMN1 gene expression was further assessed in a series of 154 astrocytomas and 22 non-neoplastic brain samples by qRT-PCR. STMN1 expression was significantly increased in malignant diffusely infiltrative astrocytomas compared with pilocytic astrocytoma (p < 0.0001). A strong correlation between MELK and STMN1 expressions was observed (r = 0.741, p < 0.0001) in glioblastoma (GBM) samples. However, no difference on survival times was found when compared GBM cases with upregulated and downregulated STMN1 (Breslow = 0.092, median survival time: 11 and 13 months, respectively). Functional assays knocking down MELK by siRNA in GBM cell line showed that gene and protein expression of both MELK and stathmin were diminished. On the other hand, when the same analysis was performed for STMN1, only stathmin gene and protein was silenced. CONCLUSIONS: The results presented herein point stahtmin as a downstream target in the MELK pathway that plays a role in malignant progression of astrocytomas.

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development.

MELK is a serine/threonine kinase involved in several cell processes, including the cell cycle, proliferation, apoptosis and mRNA processing. However, its function remains elusive. Here, we explored its role in the Xenopus early embryo and show by knockdown that xMELK (Xenopus MELK) is necessary for completion of cell division. Consistent with a role in cell division, endogenous xMELK accumulates at the equatorial cortex of anaphase blastomeres. Its relocalization is highly dynamic and correlates with a conformational rearrangement in xMELK. Overexpression of xMELK leads to failure of cytokinesis and impairs accumulation at the division furrow of activated RhoA - a pivotal regulator of cytokinesis. Furthermore, endogenous xMELK associates and colocalizes with the cytokinesis organizer anillin. Unexpectedly, our study reveals a transition in the mode of cytokinesis correlated to cell size and that implicates xMELK. Collectively, our findings disclose the importance of xMELK in cytokinesis during early development and show that the mechanism of cytokinesis changes during Xenopus early development.

Maternal embryonic leucine zipper kinase is stabilized in mitosis by phosphorylation and is partially degraded upon mitotic exit.

MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

Role of Cdc6 During Oogenesis and Early Embryo Development in Mouse and Xenopus laevis.

Cdc6 is an important player in cell cycle regulation. It is involved in the regulation of both S-phase and M-phase. Its role during oogenesis is crucial for repression of the S-phase between the first and the second meiotic M-phases, and it also regulates, via CDK1 inhibition, the M-phase entry and exit. This is of special importance for the reactivation of the major M-phase-regulating kinase CDK1 (Cyclin-Dependent Kinase 1) in oocytes entering metaphase II of meiosis and in embryo cleavage divisions, in which precise timing allows coordination between cell cycle events and developmental program of the embryo. In this chapter, we discuss the role of Cdc6 protein in oocytes and early embryos.

Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos.

Epithelia represent a unique situation where polarized cells must maintain sufficiently strong cell-cell contacts to guarantee the epithelial integrity indispensable for barrier functions. Nevertheless, epithelia must also keep sufficient plasticity which is crucial during development and morphogenesis. Adherens junctions and mechanical forces produced by the actomyosin cytoskeleton are major players for epithelial integrity maintenance and plasticity regulations. To understand how the epithelium is able to meet such a challenge, it is indispensable to determine how cellular junctions and mechanical forces acting at adherens junctions are regulated. Here, we investigate the tensile forces acting on adherens junctions via cadherin during cell division in the Xenopus embryos epithelium. Using the recently developed E-cadherin FRET tension sensor and a fastFLIM prototype microscope, we were able to measure mechanical forces applied on cadherin at cell-cell junctions. We have shown that the Xenopus epithelium is under tension, approximately 3 pN which remains stable, indicating that tensile forces acting on cadherin at the adherens junction are at equilibrium. Unexpectedly, mechanical tension across cadherin was similar between dividing and non-dividing epithelial cells.

Flexibility vs. robustness in cell cycle regulation of timing of M-phase entry in Xenopus laevis embryo cell-free extract.

During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of substrates triggering M-phase entry. Most of these substrates are dephosphorylated by PP2A. To allow phosphorylation of CDK1 substrates, PP2A is progressively inactivated upon M-phase entry. We have shown previously that the interplay between these two activities determines the timing of M-phase entry. Slight diminution of CDK1 activity by the RO3306 inhibitor delays M-phase entry in a dose-dependent manner in Xenopus embryo cell-free extract, while reduction of PP2A activity by OA inhibitor accelerates this process also in a dose-dependent manner. However, when a mixture of RO3306 and OA is added to the extract, an intermediate timing of M-phase entry is observed. Here we use a mathematical model to describe and understand this interplay. Simulations showing acceleration and delay in M-phase entry match previously described experimental data. CDC25 phosphatase is a major activator of CDK1 and acts through CDK1 Tyr15 and Thr14 dephosphorylation. Addition of CDC25 activity to our mathematical model was also consistent with our experimental results. To verify whether our assumption that the dynamics of CDC25 activation used in this model are the same in all experimental variants, we analyzed the dynamics of CDC25 phosphorylation, which reflect its activation. We confirm that these dynamics are indeed very similar in control extracts and when RO3306 and OA are present separately. However, when RO3306 and OA are added simultaneously to the extract, activation of CDC25 is slightly delayed. Integration of this parameter allowed us to improve our model. Furthermore, the pattern of CDK1 dephosphorylation on Tyr15 showed that the real dynamics of CDK1 activation are very similar in all experimental variants. The model presented here accurately describes, in mathematical terms, how the interplay between CDK1, PP2A and CDC25 controls the flexible timing of M-phase entry.

Human pEg3 kinase associates with and phosphorylates CDC25B phosphatase: a potential role for pEg3 in cell cycle regulation.

The pEg3 protein is a member of the evolutionarily conserved KIN1/PAR-1/MARK kinase family which is involved in cell polarity and microtubule dynamics. In Xenopus, pEg3 has been shown to be a cell cycle dependent kinase whose activity increases to a maximum level during mitosis of the first embryonic cell division. CDC25B is one of the three CDC25 phosphatase genes identified in human. It is thought to regulate the G2/M progression by dephosphorylating and activating the CDK/cyclin complexes. In the present study we show that the human pEg3 kinase is able to specifically phosphorylate CDC25B in vitro. One phosphorylation site was identified and corresponded to serine 323. This residue is equivalent to serine 216 in human CDC25C which plays an important role in the regulation of phosphatase during the cell cycle and at the G2 checkpoint. pEg3 is also able to specifically associate with CDC25B in vitro and in vivo. We show that the ectopic expression of active pEg3 in human U2OS cells induces an accumulation of cells in G2. This effect is counteracted by overexpression of CDC25B. Taken together these results suggest that pEg3 is a potential regulator of the G2/M progression and may act antagonistically to the CDC25B phosphatase.

PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.

During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis.

Epithelial cell division in the Xenopus laevis embryo during gastrulation.

How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

Control of timing of embryonic M-phase entry and exit is differentially sensitive to CDK1 and PP2A balance.

Harmonious embryo development requires precise coordination between the timing of the cell cycle and the developmental program. Cyclin accumulation determines the timing of the cell cycle M-phase entry and its degradation determines the timing of the M-phase exit. It is well known that CDK1 and PP2A also govern M-phase entry. However, it is unknown how this kinase and phosphatase regulate the precise timing of events at the beginning of the M-phase and how they cooperate with cyclin metabolism. Here we use Xenopus laevis one-cell embryo cell-free extract experiments to answer this question critical for understanding the regulation of embryo development. Using, separately, low concentrations of the chemical inhibitor of CDK1, RO3306 (RO), or the inhibitor of phosphatases, okadaic acid (OA), we show that moderately diminished CDK1 or PP2A activities results in a delay and an acceleration respectively, of M-phase entry. Simultaneous diminution of CDK1 and PP2A activities results in an intermediate timing of M-phase entry, prolongs the duration of M-phase and diminishes the dynamics of cyclin B2 degradation. We thus show, for the first time, that equilibrium between CDK1 and PP2A specifies the timing of M-phase entry and exit and regulates the dynamics of cyclin B degradation upon M-phase exit in Xenopus laevis first embryonic mitosis.

Cell-cycle dependent localization of MELK and its new partner RACK1 in epithelial versus mesenchyme-like cells in Xenopus embryo.

Maternal Embryonic Leucine zipper Kinase (MELK) was recently shown to be involved in cell division of Xenopus embryo epithelial cells. The cytokinetic furrow of these cells ingresses asymmetrically and is developmentally regulated. Two subpopulations of xMELK, the mMELK (for "mitotic" xMELK) and iMELK ("interphase" xMELK), which differ in their spatial and temporal regulation, are detected in Xenopus embryo. How cells regulate these two xMELK populations is unknown. In this study we show that, in epithelial cells, xMELK is present at a higher concentration at the apical junctional complex, in contrast to mesenchyme-like cells, which have uniform distribution of cortical MELK. Interestingly, mMELK and iMELK also differ by their requirements towards cell-cell contacts to establish their proper cortical localization both in epithelial and mesenchyme-like cells. Receptor for Activated protein Kinase C (RACK1), which we identified as an xMELK partner, co-localizes with xMELK at the tight junction. Moreover, a truncated RACK1 construct interferes with iMELK localization at cell-cell contacts. Collectively, our results suggest that iMELK and RACK1 are present in the same complex and that RACK1 is involved in the specific recruitment of iMELK at the apical junctional complex in epithelial cells of Xenopus embryos.