Rab-domain dynamics in endocytic membrane trafficking

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways

\u3ci\u3eAcrobasis\u3c/i\u3e Shoot Moth (Lepidoptera: Pyralidae) Infestation-Tree Height Link in a Young Black Walnut Plantation

Acrobasis shoot moth infestations were evaluated in a young black walnut progeny test for 4 years, from ages 3 to 6. Infestation levels were greatest on the largest trees in the fourth and fifth year after plantation establishment, and were declining by the sixth year. Acrobasis infestation appears to be a problem primarily on young trees less than 2.5 m in height. There was no evidence for genetic resistance to Acrobasis infestation in black walnut

A Dynamically Diluted Alignment Model Reveals the Impact of Cell Turnover on the Plasticity of Tissue Polarity Patterns

The polarisation of cells and tissues is fundamental for tissue morphogenesis during biological development and regeneration. A deeper understanding of biological polarity pattern formation can be gained from the consideration of pattern reorganisation in response to an opposing instructive cue, which we here consider by example of experimentally inducible body axis inversions in planarian flatworms. Our dynamically diluted alignment model represents three processes: entrainment of cell polarity by a global signal, local cell-cell coupling aligning polarity among neighbours and cell turnover inserting initially unpolarised cells. We show that a persistent global orienting signal determines the final mean polarity orientation in this stochastic model. Combining numerical and analytical approaches, we find that neighbour coupling retards polarity pattern reorganisation, whereas cell turnover accelerates it. We derive a formula for an effective neighbour coupling strength integrating both effects and find that the time of polarity reorganisation depends linearly on this effective parameter and no abrupt transitions are observed. This allows to determine neighbour coupling strengths from experimental observations. Our model is related to a dynamic $8$-Potts model with annealed site-dilution and makes testable predictions regarding the polarisation of dynamic systems, such as the planarian epithelium.Comment: Preprint as prior to first submission to Journal of the Royal Society Interface. 25 pages, 6 figures, plus supplement (18 pages, contains 1 table and 7 figures). A supplementary movie is available from https://dx.doi.org/10.6084/m9.figshare.c388781

Shape mode analysis exposes movement patterns in biology: flagella and flatworms as case studies

We illustrate shape mode analysis as a simple, yet powerful technique to concisely describe complex biological shapes and their dynamics. We characterize undulatory bending waves of beating flagella and reconstruct a limit cycle of flagellar oscillations, paying particular attention to the periodicity of angular data. As a second example, we analyze non-convex boundary outlines of gliding flatworms, which allows us to expose stereotypic body postures that can be related to two different locomotion mechanisms. Further, shape mode analysis based on principal component analysis allows to discriminate different flatworm species, despite large motion-associated shape variability. Thus, complex shape dynamics is characterized by a small number of shape scores that change in time. We present this method using descriptive examples, explaining abstract mathematics in a graphic way.Comment: 20 pages, 6 figures, accepted for publication in PLoS On

Treatment of the Enlarged Clitoris

Management of the enlarged clitoris, because of its import for sexual function, has been and remains one of the most controversial topics in pediatric urology. Early controversy surrounding clitoroplasty resulted from many factors including an incomplete understanding of clitoral anatomy and incorrect assumptions of the role of the clitoris in sexual function. With a better understanding of anatomy and function, procedures have evolved to preserve clitoral tissue, especially with respect to the neurovascular bundles. These changes have been made in an effort to preserve clitoral sensation and preserve orgasmic potential. It is the goal of this manuscript to describe the different procedures that have been developed for the surgical management of clitoromegally, with emphasis on the risks and benefits of each. Equally important to any discussion of such a sensitive topic is an understanding of long-term patient outcomes. As we will see, despite its importance, there has been a dearth of data in this regard. Future work in the arena of patient satisfaction will undoubtedly play a major role in directing our surgical approach

Scaling and regeneration of self-organized patterns

Biological patterns generated during development and regeneration often scale with organism size. Some organisms, e.g., flatworms, can regenerate a rescaled body plan from tissue fragments of varying sizes. Inspired by these examples, we introduce a generalization of Turing patterns that is self-organized and self-scaling. A feedback loop involving diffusing expander molecules regulates the reaction rates of a Turing system, thereby adjusting pattern length scales proportional to system size. Our model captures essential features of body plan regeneration in flatworms as observed in experiments.Comment: 5 pages, 3 color figure

Femtosecond study of the interplay between excitons, trions, and carriers in (Cd,Mn)Te quantum wells

We present an absorption study of the neutral and positively charged exciton (trion) under the influence of a femtosecond, circularly polarized, resonant pump pulse. Three populations are involved: free holes, excitons, and trions, all exhibiting transient spin polarization. In particular, a polarization of the hole gas is created by the formation of trions. The evolution of these populations is studied, including the spin flip and trion formation processes. The contributions of several mechanisms to intensity changes are evaluated, including phase space filling and spin-dependent screening. We propose a new explanation of the oscillator strength stealing phenomena observed in p-doped quantum wells, based on the screening of neutral excitons by charge carriers. We have also found that binding heavy holes into charged excitons excludes them from the interaction with the rest of the system, so that oscillator strength stealing is partially blockedComment: 4 pages, 4 figure