Cellular senescence promotes progenitor cell expansion during axolotl limb regeneration.

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure that nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact their microenvironment via Wnt pathway modulation. Further, we identify a link between Wnt signaling and senescence induction and propose that bSC-derived Wnt signals facilitate the proliferation of neighboring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in the regeneration of complex structures.

Identification of proteins in the adhesive trails of the diatom Amphora coffeaeformis.

Throughout all kingdoms of life, a large number of adhesive biomolecules have evolved to allow organisms to adhere to surfaces underwater. Proteins play an important role in the adhesion of numerous marine invertebrates (e.g. mussels, sea stars, sea urchins) whereas much less is known about the biological adhesives from marine plants, including the diatoms. Diatoms are unicellular microalgae that together with bacteria dominate marine biofilms in sunlit habitats. In this study we present the first proteomics analyses of the diatom adhesive material isolated from the tenacious fouling species Amphora coffeaeformis. We identified 21 proteins, of which 13 are diatom-specific. Ten of these proteins share a conserved C-terminal domain, termed GDPH domain, which is widespread yet not ubiquitously present in all diatom classes. Immunofluorescence localization of a GDPH domain bearing protein (Ac629) as well as two other proteins identified in this study (Ac1442, Ac9617) demonstrated that these are components of the adhesive trails that are secreted by cells that glide on surfaces. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies.

Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin.

Stability of proICA512/IA-2 and its targeting to insulin secretory granules require ß4-sheet-mediated dimerization of its ectodomain in the endoplasmic reticulum.

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that ß2- or ß4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 ß2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 ß4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-ΔNTF G553D mutant to exit the endoplasmic reticulum, and ICA512-ΔNTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 ß2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 ß4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules.

Axon regeneration genes identified by RNAi screening in C. elegans.

Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/ß-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of ß-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention.

Studying peripheral sympathetic nervous system development and neuroblastoma in zebrafish.

The combined experimental attributes of the zebrafish model system, which accommodates cellular, molecular, and genetic approaches, make it particularly well-suited for determining the mechanisms underlying normal vertebrate development as well as disease states, such as cancer. In this chapter, we describe the advantages of the zebrafish system for identifying genes and their functions that participate in the regulation of the development of the peripheral sympathetic nervous system (PSNS). The zebrafish model is a powerful system for identifying new genes and pathways that regulate PSNS development, which can then be used to genetically dissect PSNS developmental processes, such as tissue size and cell numbers, which in the past haves proved difficult to study by mutational analysis in vivo. We provide a brief review of our current understanding of genetic pathways important in PSNS development, the rationale for developing a zebrafish model, and the current knowledge of zebrafish PSNS development. Finally, we postulate that knowledge of the genes responsible for normal PSNS development in the zebrafish will help in the identification of molecular pathways that are dysfunctional in neuroblastoma, a highly malignant cancer of the PSNS.

Alterations of the cytoskeleton in all three embryonic lineages contribute to the epiboly defect of Pou5f1/Oct4 deficient MZspg zebrafish embryos.

Pou5f1/Oct4 is a transcription factor required for pluripotency of embryonic stem cells in mammals. Zebrafish pou5f1 deficient maternal and zygotic spiel ohne grenzen (MZspg) mutant embryos develop severe gastrulation defects, are dorsalized, and defective in endoderm formation. Here we analyze in detail gastrulation defects, which are manifested by a severe delay in epiboly progression. All three embryonic lineages in MZspg embryos behave abnormally during epiboly: the yolk cell forms an altered array of cortical microtubules and F-Actin, with large patches of microtubule free areas; the enveloping layer (EVL) is delayed in the coordinated cell shape changes of marginal cells, that may be mediated by F-Actin; the deep layer cells (DEL), forming the embryo proper, are non-autonomously affected in their motility and do not enter the space opening by epiboly of the EVL. Analysis of adhesiveness as well as high resolution in vivo time lapse image analysis of DEL cells suggests changed adhesive properties and inability to migrate properly on EVL and yolk syncytial layer (YSL) surfaces. Our data further reveal that during epiboly the EVL may actively probe the YSL by filopodia formation, rather than just being passively pulled vegetalwards. Our findings on the effect of Pou5f1 on cell behavior may be relevant to understand stem cell behavior and tumorigenesis involving Pou5f1.