A RhoGEF and Rho family GTPase-activating protein complex links the contractile ring to cortical microtubules at the onset of cytokinesis

The mechanism that positions the cytokinetic contractile ring is unknown, but derives from the spindle midzone. We show that an interaction between the Rho GTP exchange factor, Pebble, and the Rho family GTPase-activating protein, RacGAP50C, connects the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. We propose that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis.     

The construction of a scientific model: Otto Warburg and the building block strategy

In the years 1919 to 1923, Otto Warburg published four papers that were to revolutionise the field of photosynthesis. In these articles, he introduced a number of new techniques to measure the rate of photosynthesis, put forward a new model of the mechanism and added a completely new perspective to the topic by attempting to establish the process’s efficiency in terms of the light quantum requirement. In this paper I trace the roots of Warburg’s series of contributions to photosynthesis research by exploring three different contexts of inspiration: Warburg’s own research into cell respiration, his father’s work on the quantum yield of photochemical reactions in general and the photosynthesis work carried out by Richard Willstätter and Arthur Stoll. When these influences are considered together, it becomes clear that Warburg implemented a Building Block Strategy in his research: rather than inventing his photosynthesis model from scratch, he availed himself of fragments from other contexts, which he then recombined in a new and innovative way. This way of working is considered to be standard practice in scientific research.     

Studies on the molecular evolution of the Crocodylia: footprints in the sands of time

A reasonably large number of studies focusing on the molecular evolution of crocodilians have been completed during the past 100 years. Proteins were initially studied before DNA was known to carry the genetic information of cells and organisms, and were subsequently studied to infer changes at the DNA level. More recently, studies on the DNA itself have been completed. We have had the pleasure of taking part in or facilitating many studies conducted over the past 50 years, especially several of the earliest studies done using newly developed molecular techniques. We provide a review of the molecular genetic studies on crocodilians, summarizing the findings of these studies as well as the context in which they were undertaken. This review is a personal look at the history of molecular studies on the evolutionary biology of crocodilians. Our excuse for this focus is that our professors, our students and we have had the opportunity to be among the first to apply many new techniques to studies of crocodilians since 1950, when one of us (HCD) was a graduate student of Roland Coulson and Tom Hernandez. Although we will review much of the material in this subject area, we do not claim that it is complete. Instead, we focus our presentation on work in which we have participated or with which we are particularly familiar. We especially focus on materials relevant to the research presented at the 2(nd) International Crocodilian DNA Workshop, 7-9 November, 2001, at the San Diego Zoo. Thus, the following review also stands as a tribute to our mentors, students, and colleagues.     

Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex

The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures.     

Proteomics applied to exercise physiology: a cutting-edge technology

Exercise research has always drawn the attention of the scientific community because it can be widely applied to sport training, health improvement, and disease prevention. For many years numerous tools have been used to investigate the several physiological adaptations induced by exercise stimuli. Nowadays a closer look at the molecular mechanisms underlying metabolic pathways and muscular and cardiovascular adaptation to exercise are among the new trends in exercise physiology research. Considering this, to further understand these adaptations as well as pathology attenuation by exercise, several studies have been conducted using molecular investigations, and this trend looks set to continue. Through enormous biotechnological advances, proteomic tools have facilitated protein analysis within complex biological samples such as plasma and tissue, commonly used in exercise research. Until now, classic proteomic tools such as one- and two-dimensional polyacrylamide gel electrophoresis have been used as standard approaches to investigate proteome modulation by exercise. Furthermore, other recently developed in gel tools such as differential gel electrophoresis (DIGE) and gel-free techniques such as the protein labeling methods (ICAT, SILAC, and iTRAQ) have empowered proteomic quantitative analysis, which may successfully benefit exercise proteomic research. However, despite the three decades of 2-DE development, neither classic nor novel proteomic tools have been convincingly explored by exercise researchers. To this end, this review gives an overview of the directions in which exercise-proteome research is moving and examines the main tools that can be used as a novel strategy in exercise physiology investigation.     

Remembering Malcolm J. Casadaban

Malcolm J. Casadaban died on 13 September 2009 from an infection and was found to have a weakened strain of the bacterium Yersinia pestis in his blood. This tragic event took the life of one of the most creative and influential geneticists of our time. In the late 1970s and ''80s, Malcolm invented novel approaches which changed the way many of us did science. Jon Beckwith, Tom Silhavy, and Olaf Schneewind have chronicled his scientific life from graduate school to his death and give us insight into Malcolm''s genius.Philip Matsumura Editor in Chief, Journal of Bacteriology     

Protein folding at single-molecule resolution

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

The ciliopathy-linked protein CCDC66 is only known for its ciliary functions. This study reveals that CCDC66 also has extensive non-ciliary functions, localizing to the spindle poles, spindle midzone, central spindle and midbody throughout cell division, where it regulates mitosis and cytokinesis by promoting microtubule nucleation and organization.     

Anillin localization suggests distinct mechanisms of division plane specification in mouse oogenic meiosis I and II

Anillin is a conserved cytokinetic ring protein implicated in actomyosin cytoskeletal organization and cytoskeletal-membrane linkage. Here we explored anillin localization in the highly asymmetric divisions of the mouse oocyte that lead to the extrusion of two polar bodies. The purposes of polar body extrusion are to reduce the chromosome complement within the egg to haploid, and to retain the majority of the egg cytoplasm for embryonic development. Anillin's proposed roles in cytokinetic ring organization suggest that it plays important roles in achieving this asymmetric division. We report that during meiotic maturation, anillin mRNA is expressed and protein levels steadily rise. In meiosis I, anillin localizes to a cortical cap overlying metaphase I spindles, and a broad ring over anaphase spindles that are perpendicular to the cortex. Anillin is excluded from the cortex of the prospective first polar body, and highly enriched in the cytokinetic ring that severs the polar body from the oocyte. In meiosis II, anillin is enriched in a cortical stripe precisely coincident with and overlying the meiotic spindle midzone. These results suggest a model in which this cortical structure contributes to spindle re-alignment in meiosis II. Thus, localization of anillin as a conserved cytokinetic ring marker illustrates that the geometry of the cytokinetic ring is distinct between the two oogenic meiotic cytokineses in mammals.     

The arts and human nature: evolutionary aesthetics and the evolutionary status of art behaviours

This essay reviews one of the most recent books in a trend of new publications proffering evolutionary theorising about aesthetics and the arts—themes within an increasing literature on aspects of human life and human nature in terms of evolutionary theory. Stephen Davies’ The Artful Species links some of our aesthetic sensibilities with our evolved human nature and critically surveys the interdisciplinary debate regarding the evolutionary status of the arts. Davies’ engaging and accessible writing succeeds in demonstrating the maturity and scope of the field and his critique is timely and unparalleled. A laudable effort, however it may have benefited from espousing a co-evolutionary model more explicitly. Moreover there may be reason to question the usefulness of the standard set of distinctions (‘adaptation’, ‘spandrel’, ‘technology’) that Davies appeals to.     

Lamessende individualpsychologie: sur le rôle de l’expérimentation psychologique dans la psychiatrie d’Emil Kraepelin (seconde partie)

Contemporary historical investigations on Emil Kraepelin’s experimental work have argued variously that the psychological experiment had either no influence on his nosology, or that it was the very precondition of the nosology, or that it skewed the nosology in favor of organic disorders. The first part of this article considered Kraepelin’s experimental research in Wilhelm Wundt’s laboratory in Leipzig during the 1880s. This second part deals with Kraepelin’s work in Heidelberg in the 1890s. It emphasizes the role of the psychological experiment in stabilizing the professional and scientific legitimacy of psychiatry vis-à-vis general medicine and jurisprudence. Above all, it argues that Kraepelin’s experimental research agenda had not just nosological aims, but also clinico-diagnostic aims. Kraepelin believed that his research would help to develop a battery of diagnostic tools that could reveal prodromal symptoms, speed up diagnostic procedures, alleviate institutional overcrowding, and both generate and stabilize psychiatric norms.     

Fashioning the Immunological Self: The Biological Individuality of F. Macfarlane Burnet

During the 1940s and 1950s, the Australian microbiologist F. Macfarlane Burnet sought a biologically plausible explanation of antibody production. In this essay, we seek to recover the conceptual pathways that Burnet followed in his immunological theorizing. In so doing, we emphasize the influence of speculations on individuality, especially those of philosopher Alfred North Whitehead; the impact of cybernetics and information theory; and the contributions of clinical research into autoimmune disease that took place in Melbourne. We point to the influence of local experimental and intellectual currents on Burnet’s work. Accordingly, this essay describes an arc distinct from most other tracings of Burnet’s conceptual development, which focus on his early bacteriophage research, his fascination with the work of Julian Huxley and other biologists in the 1920s, and his interest in North Atlantic experimental investigations in the life sciences. No doubt these too were potent influences, but they seem insufficient to explain, for example, Burnet’s sudden enthusiasm in the 1940s for immunological definitions of self and not-self. We want to demonstrate here how Burnet’s deep involvement in philosophical biology – along with attention to local clinical research – provided him with additional theoretic tools and conceptual equipment, with which to explain immune function.     

Synthetic biology advancing clinical applications

The 'omics' era, with its identification of genetic and protein components, has combined with systems biology, which provided insights into network structures, to set the stage for synthetic biology, an emerging interdisciplinary life science that uses engineering principles. By capitalizing on an iterative design cycle that involves molecular and computational biology tools to assemble functional designer devices from a comprehensive catalogue of standardized biological components with predictable functions, synthetic biology has significantly advanced our understanding of complex control dynamics that program living systems. Such insights, collected over the past decade, are priming a variety of synthetic biology-inspired biomedical applications that have the potential to revolutionize drug discovery and production technologies, as well as treatment strategies for infectious diseases and metabolic disorders.     

The history of the Purine Club: a tribute to Prof. Geoffrey Burnstock

The international purinergic scientific community has lost its pioneer. Geoffrey Burnstock, born on the 10th of May 1929 in London, died on the 2nd of June 2020, aged 91, in Melbourne (Australia). Geoff was one of the most highly regarded scientists of his generation. In the 1960s and 1970s, he developed a radical and somehow heretical new theory and opened an entire new field of science, signalling via extracellular nucleotides (the “purinergic theory”), which revolutionized our understanding of how cells communicate between each other. Initially, his unconventional theory found a lot of resistance in the scientific community. Once, one scientist even threatened to devote his entire life to disproving Burnstock’s theory. Undeterred, Geoff went further on, and continued to accumulate evidence in favour of his hypothesis, and led the field ever since. He struggled to attract new scientists to this new field of research and, in the early 1990s, due to new molecular biology techniques making it possible to isolate and identify cell surface receptors for ATP and its breakdown product adenosine, did evidence emerge that eventually convinced the doubters. The number of spontaneous obituaries and messages honouring Geoff’s memory that have appeared on specialized Journals and in the public press throughout the world since last June indicates that many people are clearly affected by his death. Besides being a rigorous, ethical and extremely brilliant scientist, Geoff was an extraordinary human being, always eager to collaborate and share data, never jealous of his findings and capable of learning things even from young people. He was known for his enthusiasm, empathy and ability to motivate young scientists and promote their careers. After the establishment of the Purine Club back in the 1990s, numerous Purine Club Chapters have been formed around the world with Geoff’s help and encouragement. He has obviously also been the inspirator and founder of our Journal, Purinergic Signalling (PUSI). For this reason, Charles Kennedy, the current Editor of the Journal, and myself thought that it might be nice to invite representatives from all known Purine Clubs to send a few notes to be published in PUSI on the history of their club and how Geoff inspired, aided or supported them. Here, I have collected all their contributions and I share with the entire purinergic community my personal memories on how the Purine Club was born and developed thanks to the invaluable mentoring of Geoffrey Burnstock. I apologize in advance if I am missing some information or forgot to mention somebody, and I strongly encourage all readers to submit memories and additional information that I shall gather for future writing. Keeping alive the history of how the field developed will be the best tribute that we can play to celebrate Geoff’s work along the years.

     

Molecular ecology of fungal entomopathogens: molecular genetic tools and their applications in population and fate studies

The power of molecular genetic techniques to address ecological research questions has opened a distinct interdisciplinary research area collectively referred to as molecular ecology. Molecular ecology combines aspects of diverse research fields like population and evolutionary genetics, as well as biodiversity, conservation biology, behavioural ecology, or species-habitat interactions. Molecular techniques detect specific DNA sequence characteristics that are used as genetic markers to discriminate individuals or taxonomic groups, for instance in analyses of population and community structures, for elucidation of phylogenetic relationships, or for the characterization and monitoring of specific strains in the environment. Here, we summarize the PCR-based molecular techniques used in molecular ecological research on fungal entomopathogens and discuss novel techniques that may have relevance to the studies of entomopathogenic fungi in the future. We discuss the flow chart of the molecular ecology approaches and we highlight some of the critical steps involved. There are still many unresolved questions in the understanding of the ecology of fungal entomopathogens. These include population characteristics and relations of genotypes and habitats as well as host-pathogen interactions. Molecular tools can provide substantial support for ecological research and offer insight into this far inaccessible systems. Application of molecular ecology approaches will stimulate and accelerate new research in the field of entomophathogen ecology.     

Cell biology, chemogenomics and chemoproteomics

The scientific techniques used in molecular biological research and drug discovery have changed dramatically over the past 10 years due to the influence of genomics, proteomics and bioinformatics. Furthermore, genomics and functional genomics are now merging into a new scientific approach called chemogenomics. Advancements in the study of molecular cell biology are dependent upon "omics" researchers realizing the importance of and using the experimental tools currently available to cell biologists. For example, novel microscopic techniques utilizing advanced computer imaging allow for the examination of live specimens in a fourth dimension, viz., time. Yet, molecular biologists have not taken full advantage of these and other traditional and novel cell biology techniques for the further advancement of genomic and proteomic-oriented research. The application of traditional and novel cellular biological techniques will enhance the science of genomics. The authors hypothesize that a stronger interdisciplinary approach must be taken between cell biology (and its closely related fields) and genomics, proteomics and bio-chemoinformatics. Since there is a lot of confusion regarding many of the "omics" definitions, this article also clarifies some of the basic terminology used in genomics, and related fields. It also reviews the current status and future potential of chemogenomics and its relationship to cell biology. The authors also discuss and expand upon the differences between chemogenomics and the relatively new term--chemoproteomics. We conclude that the advances in cell biology methods and approaches and their adoption by "omics" researchers will allow scientists to maximize our knowledge about life.     

Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity

We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis.     

Molecular organization of cytokinesis node predicts the constriction rate of the contractile ring

The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.     

Making muscle elastic: the structural basis of myomesin stretching

Skeletal and cardiac muscles are remarkable biological machines that support and move our bodies and power the rhythmic work of our lungs and hearts. As well as producing active contractile force, muscles are also passively elastic, which is essential to their performance. The origins of both active contractile and passive elastic forces can be traced to the individual proteins that make up the highly ordered structure of muscle. In this Primer, we describe the organization of sarcomeres--the structural units that produce contraction--and the nature of the proteins that make muscle elastic. In particular, we focus on an elastic protein called myomesin, whose novel modular architecture helps explain elasticity.     

Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast

We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.