Eric Rivera-Milla

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain

Abstract.Reggies (flotillins) are detergent-resistant microdomains involved in the scaffolding of large heteromeric complexes that signal across the plasma membrane. Based on the presence of an evolutionarily widespread motif, reggies/flotillins have been included within the SPFH (stomatin-prohibitin-flotillin-HflC/K) protein superfamily. To better understand the origin and evolution of reggie/flotillin structure and function, we searched databases for reggie/flotillin and SPFH-like proteins in organisms at the base and beyond the animal kingdom, and used the resulting dataset to compare their structural and functional domains. Our analysis shows that the SPFH grouping has little phylogenetic support, probably due to convergent evolution of its members. We also find that reggie/flotillin homologues are highly conserved among metazoans but are absent in plants, fungi and bacteria, where only proteins with ‘reggie-like’ domains can be found. However, despite their low sequence similarities, reggie/flotillin and ‘reggie-like’ domains appear to subserve related functions, suggesting that their basic biological role was acquired independently during evolution.

Disparate evolution of prion protein domains and the distinct origin of Doppel- and prion-related loci revealed by fish-to-mammal comparisons

Prions result from the misfolding and selective accumulation of the host‐encoded prion protein (PrP) in the brain. Despite intensive research on mammalian models, basic questions about the biological role of PrP and the evolutionary origin of prion disease remain unanswered. Following our previous identification of novel fish PrP homologues, here we generated new fish PrP sequences and performed genomic analysis to demonstrate the existence of two homologous PrP loci in bony fish, which display extensive molecular variation and are highly expressed in adult and developing fish brains. The fish PrP genomic regions contain PrP‐related loci directly downstream of each PrP locus, suggesting an independent origin of prion‐related proteins in fish and mammals. Our structural prediction analysis uncovers a conserved molecular “bauplan” for all vertebrate PrPs. The C‐ and N‐terminal protein domains have evolved independently from one another, the former having retained its basic globular structure despite high sequence divergence and the latter having undergone differential expansion‐degeneration cycles in its repetitive domains. Our evolutionary analysis redefines fundamental concepts on the functional significance of PrP domains and opens up new possibilities for the experimental analysis of prion misfolding and neurodegeneration in a non‐mammalian model like the zebrafish.

An evolutionary basis for scrapie disease: identification of a fish prion mRNA

Infectious prion proteins cause neurodegenerative disease in mammals owing to the acquisition of an aberrant conformation. We cloned a Fugu rubripes gene that encodes a structurally conserved prion protein, and found rapid rates of molecular divergence among prions from different vertebrate classes, along with molecular stasis within each class. We propose that a directional trend in the evolution of prion sequence motifs associated with pathogenesis and infectivity could account for the origin of scrapie in mammals.

PHYLOGEOGRAPHY OF OLIGORYZOMYS LONGICAUDATUS (RODENTIA: SIGMODONTINAE) IN TEMPERATE SOUTH AMERICA

Abstract Phylogeographic relationships were evaluated at the intraspecific level using nucleotide sequence data from the mitochondrial cytochrome b gene of representative specimens of “colilargo” (Oligoryzomys longicaudatus) from 31 localities, along its distributional range over a large part of the western Andes and southern Argentina. Based on approximately 1,000 base pairs (bp), we recognized a single species on both the Chilean and the Argentinean side as far as at least latitude 51°S, rejecting the subspecific distinctiveness of longicaudatus and philippi. We thus placed the latter in full synonymy with O. longicaudatus as earlier studies proposed, and enlarged its range as far as Torres del Paine, about 51°S. The occurrence of subspecies in this range is doubtful given the low sequence divergence values and the absence of significant associations between haplotypes and their geography. Additionally, we hypothesized that the entrance of this species into the Chilean side of the Andes mountains occurred through the Patagonian forests of southern Argentina, with further dispersal to the north from the south.

LMX1B is Essential for the Maintenance of Differentiated Podocytes in Adult Kidneys

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

Small molecules intercept Notch signaling and the early secretory pathway

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.

Restricted expression of reggie genes and proteins during early zebrafish development

Reggies are plasma membrane‐associated proteins and characteristic markers of lipid‐raft microdomains. They are highly conserved from flies to humans and have been implicated in axon regeneration and cell process and contact formation, possibly providing functional platforms for cell‐signaling in neurons and other cell types. We analyzed reggie mRNA and protein expression patterns during early zebrafish development. All three zebrafish genes, re‐1a, ‐2a, and ‐2b, span a considerably diverse set of expression patterns, and their proteins are induced maternally, showing ubiquitous expression at early stages. Although re‐2a mRNA can be observed in differentiating neurons in the brain, spinal cord, and neurogenic placodes, re‐2b is transcribed mainly in head mesoderm, in neural crest derivates, and along somite boundaries. re‐1a mRNA is present at high levels in expression domains that overlap with the combined expression pattern of both re‐2 genes except at the somites, where it complements the pattern of re‐2b. Immunostaining on embryos reveals reggie protein localization at the cell membrane, at cell–cell contacts, and along all early axon tracts. The early phase of reggie expression suggests a basic and ubiquitous function during the first stages of embryogenesis and into the gastrula period. Upon segmentation, a second phase of expression shows distinctly localized expression patterns, indicating tissue‐specific roles and an involvement of re‐1a/re‐2a in neural development. J. Comp. Neurol. 482:257–272, 2005.

Kcnh1 voltage-gated potassium channels are essential for early zebrafish development

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation. The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

Filogeografía de las llacas chilenas del género Thylamys (Marsupialia, Didelphidae) en base a secuencias del gen mitocondrial citocromo b

Six mouse opossum species are currently recognized for the genus Thylamys in South America, of which we hypothesized the evolutionary relationships for those located in the Andean Altiplano and canyons of the Region I of Chile, as well as for those located south of the Atacama Desert down to 37˚ S. To that goal we sequenced the cytochrome b mitochondrial gene and data were analyzed using different phylogenetic criteria such as parsimony, distance and likelihood available in the program PAUP 4.0. The different phylogenetic approaches agreed in recovering two well defined clades: one constituted by Thylamys pallidior that included specimens from the coast and the “pre-cordillera” of the Region I of Chile, and the other clade constituted by Thylamys elegans that included populations located south to the Atacama Desert. Therefore, we demonstrate that populations located in the canyons and andean areas of northern Chile belong to T. pallidior, while those located south to the Atacama desert are recognized as T. elegans, results that confirm the occurrence of two thylamyine species in Chile.