Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod- driven motility and cell-cycle progression

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Highlights? Rac1 and the Scar/WAVE complex drive pseudopod-based motility of melanoblasts ? Rac1-depleted melanoblasts move using unique actin-based stubs and not blebs ? Rac1 controls pseudopod frequency but is dispensable for pseudopod formation ? Loss of Rac1 delays melanoblast cell-cycle progression and cytokinesis     

BMP-gated cell-cycle progression drives anoikis during mesenchymal collective migration

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Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

Summary The sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 M propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G₂ phase, M phase, M/G₁ interface, and g₁ phase, respectively. The polypeptide synthesis elongation factor (EF)-1 is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.Abbreviations EF-1 elongation factor 1 - MAP microtubule-associated protein - MF microfilament - MIs mitotic indices - MT microtubule     

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana-telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.     

Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.     

Nuclear envelope influences on cell-cycle progression

The nuclear envelope is a complex double membrane system that serves as a dynamic interface between the nuclear and cytoplasmic compartments. Among its many roles is to provide an anchor for gene regulatory proteins on its nucleoplasmic surface and for the cytoskeleton on its cytoplasmic surface. Both sets of anchors are proteins called NETs (nuclear envelope transmembrane proteins), embedded respectively in the inner or outer nuclear membranes. Several lines of evidence indicate that the nuclear envelope contributes to cell-cycle regulation. These contributions come from both inner and outer nuclear membrane NETs and appear to operate through several distinct mechanisms ranging from sequestration of gene-regulatory proteins to activating kinase cascades.     

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.     

Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells

In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G(2) phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G(1) phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase.     

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4^Cdt2) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.     

Dynamic changes in sweat rates and evaporation rates through clothing during hot exposure

Dynamic changes in local sweat rates (S_w) and local evaporation rates from clothing (E_cl) have been observed during hot exposure. Four young male subjects wearing a cotton T-shirt and half shorts were exposed to 40 °C/50% for 1 h following exposure to 28 °C/50% for 30 min. Amount of water absorbed in clothing (M_sw), clothing surface temperatures (T_cl), local heat flow rates, skin temperatures, body weight, rectal temperature, S_w and E_cl were continuously measured. Upon exposure to the heat, decrease in heat gain to the skin was observed as opposed to increase in S_w, E_cl, M_sw and heat gain to the clothing surface.     

Visualizing the cell-cycle progression of endothelial cells in zebrafish

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.     

Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7

Mammalian septins are GTP-binding proteins the functions of which are not well understood. Knockdown of SEPT2, 6, and 7 causes stress fibers to disintegrate and cells to lose polarity. We now show that this phenotype is induced by nuclear accumulation of the adaptor protein NCK, as the effects can be reversed or induced by cytoplasmic or nuclear NCK, respectively. NCK is carried into the nucleus by SOCS7 (suppressor of cytokine signaling 7), which possesses nuclear import/export signals. SOCS7 interacts with septins and NCK through distinct domains. DNA damage induces actin and septin rearrangement and rapid nuclear accumulation of NCK and SOCS7. Moreover, NCK expression is essential for cell-cycle arrest. The septin-SOCS7-NCK axis intersects with the canonical DNA damage cascade downstream of ATM/ATR and is essential for p53 Ser15 phosphorylation. These data illuminate an unanticipated connection between septins, SOCS7, NCK signaling, and the DNA damage response.     

Manganese effectively supports yeast cell-cycle progression in place of calcium

Metal ion requirements for the proliferation of Saccharomyces cerevisiae were investigated. We used bis-(o-aminophenoxy)-ethane- N,N,N',N'-tetraacetic acid (BAPTA), a relatively acid tolerant chelator, to reduce the free metal ion concentrations in culture media. Chelatable metal ions were added back individually and in combination. In addition to a requirement for approximately 10 pM external free Zn2+ we found an interchangeable requirement for either 66 nM free Ca2+ or only 130 pM free Mn2+. Cells depleted of Mn2+ and Ca2+ arrested as viable cells with 2 N nuclei and tended to have very small minibuds. In the absence of added Mn2+, robust growth required approximately 60 microM total internal Ca2+. In the presence of added Mn2+, robust growth continued even when internal Ca2+ was < 3% this level. Chelator- free experiments showed that MnCl2 strongly and CaCl2 weakly restored high-temperature growth of cdc1ts strains which similarly arrest as viable cells with 2 N nuclear contents and small buds. Its much greater effectiveness compared with Ca2+ suggests that Mn2+ is likely to be a physiologic mediator of bud and nuclear development in yeast. This stands in marked contrast to a claim that Ca2+ is uniquely required for cell-cycle progression in yeast. We discuss the possibility that Mn2+ may function as an intracellular signal transducer and how this possibility relates to previous claims of Ca2+'s roles in yeast metabolism.     

MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.     

Tracking changes in Z-band organization during myofibrillogenesis with FRET imaging

There are a large number of proteins associated with Z-bands in myofibrils, but the precise arrangements of most of these proteins in Z-bands are largely unknown. Even less is known about how these arrangements change during myofibrillogenesis. We have begun to address this issue using Sensitized Emission Fluorescence Resonance Energy Transfer (SE-FRET) microscopy. Cultured skeletal muscle cells from quail embryos were transfected to express fusions of alpha-actinin, FATZ, myotilin, or telethonin with cyan and yellow fluorescent proteins in various pair wise combinations. FATZ and myotilin were selected because previous biochemical studies have suggested that they bind to alpha-actinin, the major protein of the Z-band. Telethonin was selected for its reported ability to bind FATZ. Statistical analysis of data from FRET imaging studies yield results that are in agreement with published biochemical data suggesting that FATZ and myotilin bind to alpha-actinin near its C-terminus as well as to each other and that a region near the amino-terminus of FATZ is responsible for its interaction with telethonin. In addition, our analysis has revealed changes in the arrangement of alpha-actinin and FATZ that take place during the transition as the z-bodies of premyofibrils fuse to form the Z-bands of mature myofibrils. There was no evidence for a change in the arrangement of myotilin as z-bodies transformed into Z-bands. Myotilin is one Z-band protein that does not exhibit decreased dynamics as z-bodies fuse to form Z-bands. These FRET results from living cells support a stepwise model for the assembly of myofibrils.