Cellular and Molecular Logic of Spinal Cord Development
The Francis Crick Institute, London, United Kingdom
The generation of the correct neuronal subtype at the appropriate position and time in the vertebrate neural tube is the first step in the assembly of functional neural circuits. It also represents one of the best-studied examples of embryonic pattern formation. Distinct neuronal subtypes are generated in a precise spatial order from progenitor cells arrayed along the dorsal-ventral axis of the neural tube. Underpinning this organization is a complex network of extrinsic and intrinsic factors. Particularly well understood is the mechanism that determines the generation of different neuronal subtypes in ventral regions of the spinal cord. In this region of the nervous system, the secreted protein Sonic Hedgehog (Shh) acts in graded fashion to organize the pattern of neurogenesis. This is a dynamic process in which exposure to Shh generates progenitors with successively more ventral identities. At the same time tissue growth alters the proportions of cell types and elaborates the pattern. A gene regulatory network composed of transcription factors controlled by Shh signaling play an essential role in determining the graded response of cells. Thus the accurate patterning of the neural tube and the specification of neuronal subtype identity this region relies on the continuous processing and constant refinement of the cellular response to graded Shh signaling.
Identification of context-dependent tumor suppressor genes using genetic models of cancer
Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark
The transformation of normal human cells into cancer cells is recognized as a multi-step process in which cells accumulate independent genetic or epigenetic alterations that drive their progression toward a malignant phenotype. For many tumor types, specific mutations have been identified as potent cancer drivers, with well-defined roles in disease. Most tumors carry hundreds of mutations and the spectrum of mutation varies from patient to patient, and even within different parts of the same tumor. The methods used to identify cancer genes favor those with large individual effects that stand out from the ‘background noise’ of the mutational landscape in individual cancers. Evidence is emerging that ‘smaller-effect’ mutations can contribute to disease in a context-dependent manner, cooperating with other mutations. Mutations in these genes are expected to have limited effects on their own, but they can significantly potentiate the effects of other cancer drivers. However, without additional information, it is difficult to determine whether any given genetic alteration is innocuous or if it plays a role in disease. To address this issue, our laboratory has developed in vivo genetic tumor formation models to identify genes that cooperate with the established cancer driver mutations. The Drosophila tumor model allows us to screen for genes that can drive an otherwise benign tumor though malignant transformation and into metastasis – in situ. Although use of an invertebrate cancer model may seem distantly removed from human disease, it provides an opportunity to screen the entire genome for genes that can cooperate with known cancer drivers to produce malignant cancer and metastasis in the living animal. These studies have begun to identify new tumor suppressor genes and the mechanisms by which their human counterparts contribute to cancer.
Cell biology of signalling
Dunn School of Pathology, University of Oxford, United Kingdom
Signalling between cells controls most biological functions in animals. Geneticists and biochemists both tend to represent signalling pathways as diagrams where arrows connect molecular components. Yet signalling doesn’t occur in abstract space but within and between cells, so the cell biology of they systems needs to be considered. Our work on the interface of signalling and cell biology started with Drosophila genetics but is increasingly focused on the cell biology of proteins of the rhomboid-like superfamily. Rhomboids are intramembrane serine proteases that regulate a variety of signalling events. They are members of a wider rhomboid-like clan, which includes a large number of proteins that have lost the ancestral protease activity, including iRhoms. We have studied the iRhoms, which are located primarily in the endoplasmic reticulum, and have found that both in flies and mice they control signalling and trafficking. We are currently investigating the mechanism of iRhom control of trafficking, and their role in regulating inflammatory and growth factor signalling in mammals. Overall, our work emphasises the need to understand intercellular communication in the context of the cells that send and receive the signals.
Filaments of the Bacterial Cytoskeleton
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Most prokaryotic organisms are now known to contain protein filaments, constituting the Bacterial Cytoskeleton. Like their eukaryotic counterparts, the filaments participate in cellular functions such as cell division, cell shape maintenance and DNA segregation but evolutionary distances are large and the same types of filaments tend to perform different tasks in different kingdoms and organisms.
We investigate cytoskeletal systems using biochemistry, fluorescence microscopy, whole cell electron tomography, electron cryomicroscopy and X-ray crystallography, combining atomic, molecular and cell biological insights.
I will highlight how some filaments contribute to bacterial cell division, cell shape and plasmid segregation:
FtsZ is the bacterial tubulin homologue and forms a contracting ring at the division site, leading to the separation into two daughter cells. The process requires synchronisation with cell wall synthesis and involves an elusive trans-membrane complex, the divisome. FtsZ forms GTP-dependent filaments and is bound to the cell's membrane via FtsA, an actin homologue. We have reconstituted membrane constriction with only FtsZ and FtsA in vitro and have analysed the resulting filament arrangements, leading to a model of constriction and force generation.
MreB is the bacterial actin homologue, present exclusively in non-round cells. MreB is involved in cell shape maintenance through a divisome-related complex termed the elongasome. MreB and elongasome somehow direct or are directed by cell wall synthesis, leading to various cell shapes. MreB filaments are double antiparallel and bind to membrane directly. When reconstituted into liposomes, MreB filaments cause liposome deformation into rods and a model is presented how curvature selection by MreB filaments leads to rod shape in cells.
ParM is a plasmid-encoded actin-like protein that is involved in low copy number plasmid segregation through a bipolar spindle that pushes plasmids to the cell poles. We have found that ParM filaments form antiparallel spindles through direct interaction of two double helical filaments, stabilising with the overlap the otherwise dynamically unstable ends. The growing ends are protected from disassembly by the ParRC complex that links the filaments to the plasmid's centromeric region. The system is self-organising and force-generating bipolar spindles can be reconstituted and studied in vitro by TIRF microscopy.
When 1+1 makes 1: Signaling focalization promotes cell-cell fusion in yeast
Dept. of Fundamental Microbiology, Biophore, University of Lausanne, Switzerland
Cell fusion is a ubiquitous, still poorly understood process in eukaryotes, underlying fertilization and development. Yeast cells fuse during sexual reproduction to form a diploid zygote. The process relies on pheromone signaling, which arrests division and promotes sexual differentiation including polarized growth for cell pairing. Cell fusion depends on the assembly of a dedicated actin structure, the fusion focus, which serves to focalize cell wall degradation enzymes at the zone of cell-cell contact. What is the signal for fusion? How are cells protected from premature cell wall degradation, which would result in catastrophic cell lysis? I will present our recent, unpublished data addressing these questions. Briefly, by constructing autocrine cells, we show that pheromone signaling is sufficient to induce the formation of a fusion focus and promote lysis-provoking fusion attempts. We further show that focalization at the fusion focus of the pheromone signaling machinery, including receptors and transporters, is both necessary and sufficient to stabilize the focus. Thus, a positive entrainment between fusion focus formation and signaling focalization promotes cell fusion. I will further discuss our findings on the mechanism protecting cells against precocious engagement of this feed-forward system.
Host-Plasmodium Interactions: a tale of sensing and being sensed
Institute for Molecular Medicine, Lisbon, Portugal
Despite renewed eradication efforts from the international community, malaria still exerts an enormous disease burden, with nearly half the planet’s population at risk of infection. Within the human host, the disease-causing Plasmodium parasites pass through two distinct lifecycle stages, each in a different cellular environment. During the liver stage, a single Plasmodium sporozoite will invade a hepatocyte, and while sheltered there, supposedly undetected by the host, gives rise to thousands of new parasites, which will go on to initiate the subsequent blood stage of infection. While only 10-20 new parasites will be generated inside an erythrocyte, consecutive cycles of cell lysis and reinfection causing a potent host response, as well as the symptoms of malaria. The host contribution to infection outcome, on both the cellular and organismal levels has recently moved to center stage. We have identified hepatocyte molecules that modulate the success of liver stage infection, and showed that distinct host factors, not just the parasite itself, drive the onset and severity of diverse malaria syndromes. Our ongoing work indicates that the web of host-Plasmodium interactions is densely woven, with liver stage-mediated innate immune system activation, host nutritional status, and an antagonistic relationship between the two parasite stages themselves all working to modulate the balance between parasite replication and human health.
The development of colour patterns in fishes: Towards an understanding of the evolution of beauty
Max Planck Institute for Developmental Biology, Tübingen, Germany
Colour patterns are prominent features of many animals and have important functions in communication such as camouflage, kin recognition and mate choice. As targets for natural as well as sexual selection, they are of high evolutionary significance. The molecular mechanisms underlying the formation of colour patterns in vertebrates are not well understood, despite their amazing beauty and variation between closely related species. Progress in the transgenic toolkit, in vivo imaging and the availability of a large collection of mutants make the zebrafish (Danio rerio) an attractive model to study vertebrate colouration. Zebrafish display golden and blue horizontal stripes by superimposed layers of yellow xanthophores, silvery or blue iridophores and black melanophores. Lineage tracing revealed the embryonic origin of the adult pigment cells and their individual cellular behaviour during formation of the striped pattern in juvenile fish. Mutant analysis indicates that direct cell contacts between all three pigment cell types are required for the formation of the pattern, and a number of cell surface molecules have been identified as mediators of these interactions. The understanding of the mechanisms that underlie colour pattern formation is an important step towards comprehending the genetic basis of variation in the evolution of bio-diversity.
Review: Singh, A. P. and Nüsslein-Volhard, C. (2015): Zebrafish stripes as a Model for Vertebrate Colour Pattern Formation. Current Biology, 25, R81-R92
Plasmepsin IX and X: new candidate targets for old antimalarial drugs
Department of Microbiology and Molecular medicine, Faculty of Medicine, University of Geneva, Switzerland
During its erythrocytic cycle, Plasmodium parasites degrade most of the host cell haemoglobin and recycle the amino acids for the biosynthesis of their own proteins. This catabolic process takes place in the acidic food vacuole of the parasite and involves a number of proteases: aspartic proteases (Plasmepsins I, II, IV and HAP), cysteine proteases (Falcipains) and metalloprotease (Falcilicin). While the food vacuole haemoglobin degrading Plasmepsins are dispensable for parasite survival, nothing is known about the Plasmepsins IX and X that are also expressed during the erythrocytic stage but restrictively to the schizont stages. PMX and PMIX genes are refractory to genetic ablation via conventional knockout strategies. Consequently we have generated a conditional knockout of PMIX in Plasmodium falciparum using DiCre-based rapamycin induced excision. Functional dissection of PMIX-iko indicates that this protease is essential for invasion however the uncleaved substrate(s) responsible for PMIX-iko phenotype are not identified yet and the role of PMX is still under scrutiny. In Toxoplasma gondii, ASP3 is a crucial Golgi protease that plays a central role in invasion and egress from infected cells. Interestingly, ASP3 clusters phylogenically with PMIX and PMX and might at least in part share similar functions. Importantly a highly potent antimalarial compound directed against aspartyl proteases acts selectively on the late stages of the erythrocytic cycle and also targets ASP3. We hypothesize that this compound, blocking schizonts’ egress is targeting either PMIX or PMX or both.
Key issues in contemporary medicine, environmental aspects and the microbiome
Department of Structural Biology, Weizmann Institute, Rehovot, Israel
Resistance to antibiotics and the spread of antibiotics metabolites are severe problem in contemporary medicine. Structures of complexes of eubacterial-ribosomes with antibiotics paralyzing them illuminated common pathways in inhibitory-actions, synergism, differentiation and resistance. Recent structures of ribosome from a multi-resistant pathogen identified features that can account for species-specific diversity in infectious-diseases susceptibility. These may lead to design of environmental-friendly degradable alongside species-specific antibiotics-drugs, thus protecting the environment alongside preserving the microbiome.