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Controlling cell proliferation is critical for organism development, tissue homeostasis, disease, and regeneration. IQGAP3 has been shown to be required for proper cell proliferation and migration, and is associated to a number of cancers. Moreover, its expression is inversely correlated with the overall survival rate in the majority of cancers. Here, we show that IQGAP3 expression is elevated in cervical cancer and that in these cancers IQGAP3 high expression is correlated with an increased lethality. Furthermore, we demonstrate that IQGAP3 is a target of YAP, a regulator of cell cycle gene expression. IQGAP3 knockdown resulted in an increased percentage of HeLa cells in S phase, delayed progression through mitosis, and caused multipolar spindle formation and consequentially aneuploidy. Protein–protein interaction studies revealed that IQGAP3 interacts with MMS19, which is known in Drosophila to permit, by competitive binding to Xpd, Cdk7 to be fully active as a Cdk-activating kinase (CAK). Notably, IQGAP3 knockdown caused decreased MMS19 protein levels and XPD knockdown partially rescued the reduced proliferation rate upon IQGAP3 knockdown. This suggests that IQGAP3 modulates the cell cycle via the MMS19/XPD/CAK axis. Thus, in addition to governing proliferation and migration, IQGAP3 is a critical regulator of mitotic progression and genome stability. Implications: Our data indicate that, while IQGAP3 inhibition might be initially effective in decreasing cancer cell proliferation, this approach harbors the risk to promote aneuploidy and, therefore, the formation of more aggressive cancers.

The heart is a complex organ consisting of a variety of different cardiomyocytes (ventricular vs. atrial, left vs. right ventricular, working vs. nodal) as well as other cell types, including endothelial cells and vascular smooth muscle cells. Pericytes, neurons, and immune cells are less abundant, yet still important. Whereas cardiomyocytes account for around 75% of the heart volume, 50-70% of the cells in the heart are non-myocytes. This complexity of the heart underlines the difficulties in interpreting data obtained in vivo. In the field of cardiac regeneration, it remains unclear whether it is possible to induce a significant number of cardiomyocytes to proliferate and whether the often-observed improvement in cardiac function after experimental therapies is due to the induction of cardiomyocyte proliferation. Therefore, the reductionist approach inherent to cultures of isolated cells continues to be of great importance, even though it is important to study heart disease in vivo due to interactions of the different cell types. Cultured cardiomyocytes allow for easy manipulation of cell behavior (e.g., cell division) and its analysis (e.g., live-cell imaging). In addition, isolated cells in culture are a valuable tool for pharmacological and toxicological studies. This chapter offers a practical guide to isolate and culture primary neonatal and adult rat cardiomyocytes and a detailed protocol for live-cell imaging of embryonic and neonatal cardiomyocytes.

Distinctly organized microtubule networks contribute to the function of differentiated cell types such as neurons, epithelial cells, skeletal myotubes, and cardiomyocytes. In striated (i.e., skeletal and cardiac) muscle cells, the nuclear envelope acts as the dominant microtubule-organizing center (MTOC) and the function of the centrosome—the canonical MTOC of mammalian cells—is attenuated, a common feature of differentiated cell types. We summarize the mechanisms known to underlie MTOC formation at the nuclear envelope, discuss the significance of the nuclear envelope MTOC for muscle function and cell cycle progression, and outline potential mechanisms of centrosome attenuation.

AimsAfter birth mammalian cardiomyocytes initiate a last cell cycle which results in binucleation due to cytokinesis failure. Despite its importance for cardiac regenerative therapies, this process is poorly understood. Here, we aimed at a better understanding of the difference between cardiomyocyte proliferation and binucleation and providing a new tool to distinguish these two processes.Methods and resultsMonitoring of cell division by time-lapse imaging revealed that rat cardiomyocyte binucleation stems from a failure to properly ingress the cleavage furrow. Astral microtubule required for actomyosin ring anchorage and thus furrow ingression were not symmetrically distributed at the periphery of the equatorial region during anaphase in binucleating cardiomyocytes. Consequently, RhoA, the master regulator of actomyosin ring formation and constriction, non-muscle myosin IIB, a central component of the actomyosin ring, as well as IQGAP3 were abnormally localized during cytokinesis. In agreement with improper furrow ingression, binucleation in vitro and in vivo was associated with a failure of RhoA and IQGAP3 to localize to the stembody of the midbody.ConclusionTaken together, these results indicate that naturally occurring cytokinesis failure in primary cardiomyocytes is due to an aberrant mitotic microtubule apparatus resulting in inefficient anchorage of the actomyosin ring to the plasma cell membrane. Thus, cardiomyocyte binucleation and division can be discriminated by the analysis of RhoA as well as IQGAP3 localization.

Background The majority of adult human, mouse and rat cardiomyocytes is not diploid mononucleated. Nevertheless, the current literature on heart regeneration based on cardiomyocyte proliferation focuses mainly on the proliferation capacity of diploid mononucleated cardiomyocytes, instead of the more abundant mononucleated polyploid or binucleated cardiomyocytes. Here, we aimed at a better understanding of the process of mitosis and cell division in postnatal binucleated cardiomyocytes. Methods and results Postnatal rat binucleated cardiomyocytes were stimulated to re-enter the cell cycle either by fetal bovine serum or a combination of fibroblast growth factor 1 and p38 MAP kinase inhibitor. Phase-contrast videos revealed that binucleated cardiomyocytes form one metaphase plate and preferentially undergo afterwards cytokinesis failure. The maximum rate of cell division of video-recorded binucleated cardiomyocytes was around 6%. Immunofluorescence analyses of centriole number in mitotic binucleated cardiomyocytes revealed that these cells contain more than four centrioles, which can be paired as well as unpaired. In agreement with multiple and/or unpaired centrioles, multipolar spindle formation was observed in mitotic binucleated cardiomyocytes using fluorescence live imaging of tubulin-GFP. Multipoles were transient and resolved into pseudo-bipolar spindles both in case of cell division and cytokinesis failure. Notably, centrioles were in most cases unevenly distributed among daughter cells. Conclusions Our results indicate that postnatal binucleated cardiomyocytes upon stimulation can enter mitosis, cope with their multiple and/or unpaired centrioles by forming pseudo-bipolar spindles, and divide.

Annals of neurology 79(5), 826-840 (2016). doi:10.1002/ana.24633, Published by Wiley-Blackwell, Hoboken, NJ

The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.

Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart. DOI: http://dx.doi.org/10.7554/eLife.05563.001, eLife digest Muscle cells in the heart contract in regular rhythms to pump blood around the body. In humans, rats and other mammals, the vast majority of heart muscle cells lose the ability to divide shortly after birth. Therefore, the heart is unable to replace cells that are lost over the life of the individual, for example, during a heart attack. If too many of these cells are lost, the heart will be unable to pump effectively, which can lead to heart failure. Currently, the only treatment option in humans with heart failure is to perform a heart transplant. Some animals, such as newts and zebrafish, are able to replace lost heart muscle cells throughout their lifetimes. Thus, these species are able to fully regenerate their hearts even after 20% has been removed. This suggests that it might be possible to manipulate human heart muscle cells to make them divide and regenerate the heart. Recent research has suggested that structures called centrosomes, known to be required to separate copies of the DNA during cell division, are used as a hub to integrate the initial signals that determine whether a cell should divide or not. Here, Zebrowski et al. studied the centrosomes of heart muscle cells in rats, newts and zebrafish. The experiments show that the centrosomes in rat heart muscle cells are dissembled shortly after birth. Centrosomes are made of several proteins and, in the rat cells, these proteins moved to the membrane that surrounded the nucleus. On the other hand, the centrosomes in the heart muscle cells of the adult newts and zebrafish remained intact. Further experiments found that that breaking apart the centrosomes of heart muscle cells taken from newborn rats stops these cells from dividing. Zebrowski et al.'s findings suggest that the loss of centrosomes after birth is a possible reason why the hearts of adult humans and other mammals are unable to regenerate after injury. In the future, these findings may aid the development of methods to regenerate human heart muscle and new treatments that may limit division of cancer cells. DOI: http://dx.doi.org/10.7554/eLife.05563.002