Jochen C. Rink

Rab Conversion as a Mechanism of Progression from Early to Late Endosomes

The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab 5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger endosomes that migrate from the cell periphery to the center where Rab 5 is rapidly replaced with Rab 7. The class C VPS/HOPS complex, an established GEF for Rab 7, interacts with Rab 5 and is required for Rab 5-to-Rab 7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late endosomes.

Systems survey of endocytosis by multiparametric image analysis

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-β and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

Formaldehyde-based whole-mount in situ hybridization method for planarians.

Whole‐mount in situ hybridization (WISH) is a powerful tool for visualizing gene expression patterns in specific cell and tissue types. Each model organism presents its own unique set of challenges for achieving robust and reproducible staining with cellular resolution. Here, we describe a formaldehyde‐based WISH method for the freshwater planarian Schmidtea mediterranea developed by systematically comparing and optimizing techniques for fixation, permeabilization, hybridization, and postprocessing. The new method gives robust, high‐resolution labeling in fine anatomical detail, allows co‐labeling with fluorescent probes, and is sufficiently sensitive to resolve the expression pattern of a microRNA in planarians. Our WISH methodology not only provides significant advancements over current protocols that make it a valuable asset for the planarian community, but should also find wide applicability in WISH methods used in other systems. Developmental Dynamics 238:443–450, 2009.

Planarian Hh signaling regulates regeneration polarity and links Hh pathway evolution to cilia.

Hedgehog, Flatworms, and Regeneration The Hedgehog (Hh) signaling pathway plays pivotal roles during embryonic development, post-embryonic tissue maintenance, and disease. In spite of having at its core a collection of highly conserved components, the Hh pathway displays unusual differences in signal transduction mechanisms among flies, fish, and mammals. Using planarians (nonparasitic flatworms), Rink et al. (p. 1406, published online 22 October) report a key role for Hh signaling during adult regeneration and provide evidence that the perplexing diversity of transduction mechanisms may have arisen from animal-specific losses of an ancestral association between cilia and Hh signaling. Analysis of the Hedgehog signaling pathway in planaria suggests an ancestral association of this signaling pathway and cilia function. The Hedgehog (Hh) signaling pathway plays multiple essential roles during metazoan development, homeostasis, and disease. Although core protein components are highly conserved, the variations in Hh signal transduction mechanisms exhibited by existing model systems (Drosophila, fish, and mammals) are difficult to understand. We characterized the Hh pathway in planarians. Hh signaling is essential for establishing the anterior/posterior axis during regeneration by modulating wnt expression. Moreover, RNA interference methods to reduce signal transduction proteins Cos2/Kif27/Kif7, Fused, or Iguana do not result in detectable Hh signaling defects; however, these proteins are essential for planarian ciliogenesis. Our study expands the understanding of Hh signaling in the animal kingdom and suggests an ancestral mechanistic link between Hh signaling and the function of cilia.

Stem cell systems and regeneration in planaria

Planarians are members of the Platyhelminthes (flatworms). These animals have evolved a remarkable stem cell system. A single pluripotent adult stem cell type (“neoblast”) gives rise to the entire range of cell types and organs in the planarian body plan, including a brain, digestive-, excretory-, sensory- and reproductive systems. Neoblasts are abundantly present throughout the mesenchyme and divide continuously. The resulting stream of progenitors and turnover of differentiated cells drive the rapid self-renewal of the entire animal within a matter of weeks. Planarians grow and literally de-grow (“shrink”) by the food supply-dependent adjustment of organismal turnover rates, scaling body plan proportions over as much as a 50-fold size range. Their dynamic body architecture further allows astonishing regenerative abilities, including the regeneration of complete and perfectly proportioned animals even from tiny tissue remnants. Planarians as an experimental system, therefore, provide unique opportunities for addressing a spectrum of current problems in stem cell research, including the evolutionary conservation of pluripotency, the dynamic organization of differentiation lineages and the mechanisms underlying organismal stem cell homeostasis. The first part of this review focuses on the molecular biology of neoblasts as pluripotent stem cells. The second part examines the fascinating mechanistic and conceptual challenges posed by a stem cell system that epitomizes a universal design principle of biological systems: the dynamic steady state.

Membrane identity and GTPase cascades regulated by toggle and cut-out switches

Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5‐to‐Rab7 conversion is rather based on a newly characterized ‘cut‐out switch’ analogous to an electrical safety‐breaker. Both designs require cooperativity of auto‐activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut‐out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut‐out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes.

Plant nuclei can contain extensive grooves and invaginations

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Reactivating head regrowth in a regeneration-deficient planarian species

Species capable of regenerating lost body parts occur throughout the animal kingdom, yet close relatives are often regeneration incompetent. Why in the face of ‘survival of the fittest’ some animals regenerate but others do not remains a fascinating question. Planarian flatworms are well known and studied for their ability to regenerate from minute tissue pieces, yet species with limited regeneration abilities have been described even amongst planarians. Here we report the characterization of the regeneration defect in the planarian Dendrocoelum lacteum and its successful rescue. Tissue fragments cut from the posterior half of the body of this species are unable to regenerate a head and ultimately die. We find that this defect originates during the early stages of head specification, which require inhibition of canonical Wnt signalling in other planarian species. Notably, RNA interference (RNAi)-mediated knockdown of Dlac-β-catenin-1, the Wnt signal transducer, restored the regeneration of fully functional heads on tail pieces, rescuing D. lacteum’s regeneration defect. Our results demonstrate the utility of comparative studies towards the reactivation of regenerative abilities in regeneration-deficient animals. Furthermore, the availability of D. lacteum as a regeneration-impaired planarian model species provides a first step towards elucidating the evolutionary mechanisms that ultimately determine why some animals regenerate and others do not.

The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling

The maintenance of organs and their regeneration in case of injury are crucial to the survival of all animals. High rates of tissue turnover and nearly unlimited regenerative capabilities make planarian flatworms an ideal system with which to investigate these important processes, yet little is known about the cell biology and anatomy of their organs. Here we focus on the planarian excretory system, which consists of internal protonephridial tubules. We find that these assemble into complex branching patterns with a stereotyped succession of cell types along their length. Organ regeneration is likely to originate from a precursor structure arising in the blastema, which undergoes extensive branching morphogenesis. In an RNAi screen of signaling molecules, we identified an EGF receptor (Smed-EGFR-5) as a crucial regulator of branching morphogenesis and maintenance. Overall, our characterization of the planarian protonephridial system establishes a new paradigm for regenerative organogenesis and provides a platform for exploring its functional and evolutionary homologies with vertebrate excretory systems.

The mid-developmental transition and the evolution of animal body plans

Animals are grouped into ~35 ‘phyla’ based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development—known as the phylotypic period—is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent ‘mid-developmental transition’ that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.