Charting the secretory pathway in a simple eukaryote

George Palade, a founding father of cell biology and of the American Society for Cell Biology (ASCB), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. His vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. My laboratory has dissected these pathways with complementary genetic and biochemical approaches. Peter Novick, one of my first graduate students, isolated secretion mutants of Saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding SEC genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. A biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize Sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (ER) membrane (Sec61) and the cytoplasmic coat protein complex (COPII) that captures cargo proteins into transport vesicles that bud from the ER.     

Workshop on "What do you need to know about doing cell biology experiments in space?"

The "What do you need to know about doing cell biology experiments in space?" workshop represented a continued international collaboration between cell culture hardware developers and scientists, partly due to the enhanced collaboration in space life sciences spurred on by the International Space Life Sciences Working Group. The workshop was organized into three sessions. The first session provided an overview of the general effects of space flight, including definition of the microgravity environment, the radiation environment, and issues surrounding mass transport. The session concluded with an important overview of using space centrifuges as Earth gravity (1-g) controls, including understanding the contribution of inertial shear forces. The second session described existing and planned hardware facilities developed to support cell culture research, ranging from small hand-held hardware to breadbox-sized Shuttle middeck hardware to complete facility racks. Hardware designed for use on the Shuttle, ISS, and in free flyers was described. The third session provided advice from experienced space flight cell biology principal investigators to new investigators in the field. This special issue of the Journal of Gravitational Physiology includes externally peer-reviewed papers from all three sessions.     

Reprogramming somatic cells to pluripotency: A fresh look at Yamanaka’s model

In 2006, Dr Shinya Yamanaka succeeded to reprogram somatic cells into pluripotent stem cells (iPSC) by delivering the genes encoding Oct4, Sox2, Klf4, and c-Myc. This achievement represents a fundamental breakthrough in stem cell biology and opens up a new era in regenerative medicine. However, the molecular processes by which somatic cells are reprogrammed into iPSC remain poorly understood. In 2009, Yamanaka proposed the elite and stochastic models for reprogramming mechanisms. To date, many investigators in the field of iPSC research support the concept of stochastic model, i.e., somatic cell reprogramming is an event of epigenetic transformation. A mathematical model, f (C_d, k), has also been proposed to predict the stochastic process. Here we wish to revisit the Yamanaka model and summarize the recent advances in this research field.     

Cell biology of stem cells: an enigma of asymmetry and self-renewal

Stem cells present a vast, new terrain of cell biology. A central question in stem cell research is how stem cells achieve asymmetric divisions to replicate themselves while producing differentiated daughter cells. This hallmark of stem cells is manifested either strictly during each mitosis or loosely among several divisions. Current research has revealed the crucial roles of niche signaling, intrinsic cell polarity, subcellular localization mechanism, asymmetric centrosomes and spindles, as well as cell cycle regulators in establishing self-renewing asymmetry during stem cell division. Much of this progress has benefited from studies in model stem cell systems such as Drosophila melanogaster neuroblasts and germline stem cells and mammalian skin stem cells. Further investigations of these questions in diverse types of stem cells will significantly advance our knowledge of cell biology and allow us to effectively harness stem cells for therapeutic applications.     

Mitotic histiocytes and intranuclear Langerhans cell granules in histiocytosis X

Several mitotic histiocytes containing Langerhans cell granules were found in the lymph nodes of Letterer-Siwe disease. Some histiocytes of Hand-Schüller-Christian disease contained Langerhans cell granules within the nuclei. These Langerhans cell granules were not in 'nuclear pseudo-inclusions,' but were freely scattered inside the nuclei. We suggest that the Langerhans cell granules may get trapped in the nucleus during mitosis, since several investigators have suggested that mitosis causes other intranuclear organelles to get trapped. We also speculate that cells containing Langerhans cell granules may divide and increase by mitosis.     

Twenty years of research on primordial germ cells.

Just twenty years ago I was preparing a research project centred on establishing methods for the isolation and culture of mouse primordial germ cells (PGCs). The project had been suggested to me by Anne McLaren and was to be developed at the Medical Research Council (MRC) "Mammalian Development Unit" in London under the direction of Anne herself. At that time I was a young postdoctoral researcher at the Institute of Histology and Embryology of the University of Rome "La Sapienza" and did not imagine that my decision to be involved in this project would signal a profound switch in my scientific life. From then on my research would mostly concentrate on primordial germ cell biology. I feel like saying that the modern history of mammalian primordial germ cells began twenty years ago at the MRC Mammalian Development Unit under Anne McLaren's impulse. It is not surprising that among the most active researchers in the last twenty years in studying mammalian primordial germ cells, three, namely Chris Wylie, Peter Donovan and myself, began their studies under Anne McLaren's guidance. Over the years, Anne's suggestions and encouragement were always precious for my studies and her presence marked my most important findings on PGC biology. She often invited me to present the results obtained in my laboratory to workshops and congresses. In the present article some of these results particularly influenced by Anne's teaching and suggestions will be briefly reviewed.     

Are Anaphase Events Really Irreversible? The Endmost Stages of Cell Division and the Paradox of the DNA Double-Strand Break Repair

It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double-strand breaks (DSBs), likely to find a donor sequence for homology-directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase.     

Melanocyte development: with a message of encouragement to young women scientists

This is a semi-biographical review describing my research on melanocyte development and related personal experiences. Having been educated and trained as a dermatologist, I have been involved in many clinically-oriented studies, however, what has always interested me the most is pigment cell biology. Since I started working at St Marianna University in 1991, I have been undertaking research on melanocyte development and relevant growth factors using mice as models. My research in this field was inspired by my collaborations with various scientists, mostly from the field of biology. Many of these specialists I have met at meetings of the Societies of Pigment Cell Research (PCR). Stem cell factor (SCF, Kitl) and endothelin 3 (EDN3) have been identified as indispensable factors regulating the development of melanocytes. Mice mutant at loci encoding those factors (or their receptors) such as Sl/Sl (receptors W/W) and ls/ls (receptors s/s) have white coat colors and white patches, respectively. Our murine neural crest cell (NCC) primary cultures derived from Sl/Sl embryos showed that EDN3 cannot develop melanocyte precursors without SCF and that EDN3 can elicit proliferation and differentiation in the presence of SCF. These results suggest that without EDN3 and the endothelin type B receptor (EDNRB), melanocytes can not fully increase in number, which could well be the cause of the partial white coat color of ls/ls and s/s mice. Contamination with factors derived from the serum in medium or in feeder cells sometimes causes experimental errors, and therefore we established three immortal cell lines derived from NCC in different developmental stages and designated them as NCCmelb4, NCCmelb4M5 and NCCmelan5, all of which can survive without feeder cells. Using these cell lines and NCC primary cultures, we studied the effect of many factors related to melanocyte development. From the results, it has become evident that Vitamin D3 induces EDNRB expression by NCCmelb4 cells. In addition to the International Pigment Cell Conference (IPCC), I have also taken part in many annual meetings of the Japanese Society for Pigment Cell Research (JSPCR), Pan American Society for Pigment Cell Research (PASPCR) and European Society for Pigment Cell Research (ESPCR). Not only have I learned a great deal, I have enjoyed myself immensely at those meetings. Moreover, I have made many good friends there, some of whom I have collaborated with in my research. To conclude, I would like to give my message 'be ambitious' to young scientists, especially young women.     

Regulation of actin dynamics and protein trafficking during spermatogenesis – Insights into a complex process

Abstract

In the mammalian testis, extensive restructuring takes place across the seminiferous epithelium at the Sertoli–Sertoli and Sertoli–germ cell interface during the epithelial cycle of spermatogenesis, which is important to facilitate changes in the cell shape and morphology of developing germ cells. However, precise communications also take place at the cell junctions to coordinate the discrete events pertinent to spermatogenesis, namely spermatogonial renewal via mitosis, cell cycle progression and meiosis, spermiogenesis and spermiation. It is obvious that these cellular events are intimately related to the underlying actin-based cytoskeleton which is being used by different cell junctions for their attachment. However, little is known on the biology and regulation of this cytoskeleton, in particular its possible involvement in endocytic vesicle-mediated trafficking during spermatogenesis, which in turn affects cell adhesive function and communication at the cell–cell interface. Studies in other epithelia in recent years have shed insightful information on the intimate involvement of actin dynamics and protein trafficking in regulating cell adhesion and communications. The goal of this critical review is to provide an updated assessment of the latest findings in the field on how these complex processes are being regulated during spermatogenesis. We also provide a working model based on the latest findings in the field including our laboratory to provide our thoughts on an apparent complicated subject, which also serves as the framework for investigators in the field. It is obvious that this model will be rapidly updated when more data are available in future years.     

染色体浓缩素

染色体的形成是细胞周期的重要事件,然而有关染色体构筑动力学的分子机制仍未阐明。近年来对染色体浓缩素的分离与研究,为认识DNA浓缩和染色体构建机制提供了重要的线索,是细胞生物学研究领域的里程碑。现对浓缩素的发现过程,浓缩素在有丝分裂和减数分裂中的作用,浓缩素与黏着素的关系,浓缩素参与基因调节等方面进行综述,为相关领域的研究者提供参考。     

Asymmetric cell division: recent developments and their implications for tumour biology

The ability of cells to divide asymmetrically is essential for generating diverse cell types during development. The past 10 years have seen tremendous progress in our understanding of this important biological process. We have learned that localized phosphorylation events are responsible for the asymmetric segregation of cell fate determinants in mitosis and that centrosomes and microtubules play important parts in this process. The relevance of asymmetric cell division for stem cell biology has added a new dimension to the field, and exciting connections between asymmetric cell division and tumorigenesis have begun to emerge.     

Sir John Gurdon: Father of nuclear reprogramming

Sir John Gurdon founded the field of nuclear reprogramming. His work set the stage for the ever burgeoning area of stem cell biology and regenerative medicine. Here I provide personal reflections on times I shared with John Gurdon and professional reflections of the impact of his ground-breaking research on my own development as a scientist and on the field in general. His paradigm-shifting experiments will continue to provoke scientists to think outside the box for many years to come.     

The early impact of genetics on our understanding of cell cycle regulation in Aspergillus nidulans

The application of genetic analysis was crucial to the rapid progress that has been made in cell cycle research. Ron Morris, one of the first to apply genetics to cell cycle research, developed Aspergillus nidulans into an important model system for the analysis of many aspects of cell biology. Within the area of cell cycle research, Ron's laboratory is noted for development of novel cell biological and molecular genetic approaches as well as seminal insights regarding the regulation of mitosis, checkpoint regulation of the cell cycle, and the role of microtubule-based motors in chromosome segregation. In this special edition of FGB dedicated to Ron Morris, and in light of the recent progress in fungal genomics, we review the outstanding contributions his work made to our understanding of mitotic regulation. Indeed, his efforts have provided many mutants and experimental tools along with the conceptual framework for current and future studies of mitosis in A. nidulans.     

Cancer stem cells: A guide for skeptics

Cancer stem cells (CSC) were postulated to exist many years ago as cells within a tumor that regenerate the tumor following treatment. A stochastic clonal evolution model was used to explain observed tumor heterogeneity. Recently, xenotransplantation studies have demonstrated that prospectively identifiable subpopulations from human cancers can initiate tumors in immune deficient mice, and these results along with recent advances in stem cell biology have generated much excitement in the cancer field. The modern CSC theory posits a hierarchy of cells analogous to normal stem cell development. Some controversy remains, however, as to whether these tumor initiating cells truly represent CSC, and whether the modern CSC field can live up to the promise of providing improved cancer treatments based on a novel model of cancer biology. Recent data from CSC investigators are discussed critically. J. Cell. Biochem. 106: 745–749, 2009. © 2009 Wiley‐Liss, Inc.     

From imaging to understanding: Frontiers in Live Cell Imaging, Bethesda, MD, April 19-21, 2006

A recent meeting entitled Frontiers in Live Cell Imaging was attended by more than 400 cell biologists, physicists, chemists, mathematicians, and engineers. Unlike typical special topics meetings, which bring together investigators in a defined field primarily to review recent progress, the purpose of this meeting was to promote cross-disciplinary interactions by introducing emerging methods on the one hand and important biological applications on the other. The goal was to turn live cell imaging from a “technique” used in cell biology into a new exploratory science that combines a number of research fields.     

The microenvironment matters

The physical and biochemical properties of the microenvironment regulate cell behavior and modulate tissue development and homeostasis. Likewise, the physical and interpersonal cues a trainee receives profoundly influence his or her scientific development, research perspective, and future success. My cell biology career has been greatly impacted by the flavor of the scientific environments I have trained within and the diverse research mentoring I have received. Interactions with physical and life scientists and trainees and exposure to a diverse assortment of interdisciplinary environments have and continue to shape my research vision, guide my experimental trajectory, and contribute to my scientific success and personal happiness.     

Using the Drosophila melanogaster D17-c3 cell culture system to study cell motility

Cultured Drosophila melanogaster S2 and S2R+ cell lines have become important tools for uncovering fundamental aspects of cell biology as well as for gene discovery. Despite their utility, these cell lines are nonmotile and cannot build polarized structures or cell-cell contacts. Here we outline a previously isolated, but uncharacterized, Drosophila cell line named Dm-D17-c3 (or D17). These cells spread and migrate in culture, form cell-cell junctions and are susceptible to RNA interference (RNAi). Using this protocol, we describe how investigators, upon receiving cells from the Bloomington stock center, can culture cells and prepare the necessary reagents to plate and image migrating D17 cells; they can then be used to examine intracellular dynamics or observe loss-of-function RNAi phenotypes using an in vitro scratch or wound healing assay. From first thawing frozen ampules of D17 cells, investigators can expect to begin assaying RNAi phenotypes in D17 cells within roughly 2-3 weeks.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Evolutionarily conserved concepts in glial cell biology

The evolutionary conservation of glial cells has been appreciated since Ramon y Cajal and Del Rio Hortega first described the morphological features of cells in the nervous system. We now appreciate that glial cells have essential roles throughout life in most nervous systems. The field of glial cell biology has grown exponentially in the last ten years. This new wealth of knowledge has been aided by seminal findings in non-mammalian model systems. Ultimately, such concepts help us to understand glia in mammalian nervous systems. Rather than summarizing the field of glial biology, I will first briefly introduce glia in non-mammalian models systems. Then, highlight seminal findings across the glial field that utilized non-mammalian model systems to advance our understanding of the mammalian nervous system. Finally, I will call attention to some recent findings that introduce new questions about glial cell biology that will be investigated for years to come.