Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 140
Filtrar
1.
Sci Rep ; 12(1): 5829, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35388060

RESUMO

The evolution of Australia's distinctive marsupial fauna has long been linked to the onset of continent-wide aridity. However, how this profound climate change event affected the diversification of extant lineages is still hotly debated. Here, we assemble a DNA sequence dataset of Macropodoidea-the clade comprising kangaroos and their relatives-that incorporates a complete mitogenome for the Desert 'rat-kangaroo', Caloprymnus campestris. This enigmatic species went extinct nearly 90 years ago and is known from a handful of museum specimens. Caloprymnus is significant because it was the only macropodoid restricted to extreme desert environments, and therefore calibrates the group's specialisation for increasingly arid conditions. Our robustly supported phylogenies nest Caloprymnus amongst the bettongs Aepyprymnus and Bettongia. Dated ancestral range estimations further reveal that the Caloprymnus-Bettongia lineage originated in nascent xeric settings during the middle to late Miocene, ~ 12 million years ago (Ma), but subsequently radiated into fragmenting mesic habitats after the Pliocene to mid-Pleistocene. This timeframe parallels the ancestral divergences of kangaroos in woodlands and forests, but predates their adaptive dispersal into proliferating dry shrublands and grasslands from the late Miocene to mid-Pleistocene, after ~ 7 Ma. We thus demonstrate that protracted changes in both climate and vegetation likely staged the emergence of modern arid zone macropodoids.


Assuntos
Genoma Mitocondrial , Macropodidae , Adaptação Fisiológica , Animais , Clima Desértico , Filogenia , Potoroidae , Ratos
2.
Sci Rep ; 11(1): 17775, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493783

RESUMO

Macropod progressive periodontal disease (MPPD) is a necrotizing, polymicrobial, inflammatory disease commonly diagnosed in captive macropods. MPPD is characterized by gingivitis associated with dental plaque formation, which progresses to periodontitis and then to osteomyelitis of the mandible or maxilla. However, the underlying microbial causes of this disease remain poorly understood. In this study, we collected 27 oral plaque samples and associated clinical records from 22 captive Macropodidae and Potoroidae individuals that were undergoing clinical examination at Adelaide and Monarto Zoos in South Australia (15 healthy, 7 gingivitis and 5 periodontitis-osteomyelitis samples). The V3-V4 region of the 16S ribosomal RNA gene was sequenced using an Illumina Miseq to explore links between MPPD and oral bacteria in these animals. Compositional differences were detected between the microbiota of periodontitis-osteomyelitis cases compared to healthy samples (p-value with Bonferroni correction < 0.01), as well as gingivitis cases compared to healthy samples (p-value with Bonferroni correction < 0.05) using Permutational Multivariate Analysis of Variance (PERMANOVA). An overabundance of Porphyromonas, Fusobacterium, and Bacteroides taxa was also identified in animals with MPPD compared to healthy individuals using linear discriminant analysis effect size (LEfSe; p = < 0.05). An increased abundance of Desulfomicrobium also was detected in MPPD samples (LEfSe; p < 0.05), which could potentially reflect differences in disease progression. This is the first microbiota analysis of MPPD in captive macropods, and these results support a polymicrobial pathogenesis of MPPD, suggesting that the microbial interactions underpinning MPPD may be more complex than previously documented.


Assuntos
Bacteroides/isolamento & purificação , Placa Dentária/veterinária , Fusobacterium/isolamento & purificação , Gengivite/veterinária , Macropodidae/microbiologia , Microbiota , Periodontite/veterinária , Porphyromonas/isolamento & purificação , Potoroidae/microbiologia , Animais , Animais de Zoológico/microbiologia , Biodiversidade , Coinfecção , Placa Dentária/microbiologia , Progressão da Doença , Gengivite/microbiologia , Doenças Mandibulares/microbiologia , Doenças Mandibulares/veterinária , Doenças Maxilares/microbiologia , Doenças Maxilares/veterinária , Osteomielite/microbiologia , Osteomielite/veterinária , Periodontite/microbiologia , Austrália do Sul
3.
Angew Chem Int Ed Engl ; 60(43): 23212-23216, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34415670

RESUMO

Recently described rhizolutin and collinolactone isolated from Streptomyces Gö 40/10 share the same novel carbon scaffold. Analyses by NMR and X-Ray crystallography verify the structure of collinolactone and propose a revision of rhizolutin's stereochemistry. Isotope-labeled precursor feeding shows that collinolactone is biosynthesized via type I polyketide synthase with Baeyer-Villiger oxidation. CRISPR-based genetic strategies led to the identification of the biosynthetic gene cluster and a high-production strain. Chemical semisyntheses yielded collinolactone analogues with inhibitory effects on L929 cell line. Fluorescence microscopy revealed that only particular analogues induce monopolar spindles impairing cell division in mitosis. Inspired by the Alzheimer-protective activity of rhizolutin, we investigated the neuroprotective effects of collinolactone and its analogues on glutamate-sensitive cells (HT22) and indeed, natural collinolactone displays distinct neuroprotection from intracellular oxidative stress.


Assuntos
Diterpenos/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Linhagem Celular , Diterpenos/química , Diterpenos/metabolismo , Camundongos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Potoroidae , Fuso Acromático/efeitos dos fármacos
4.
J Wildl Dis ; 57(3): 515-524, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33901289

RESUMO

Over two field seasons during 2014-15, 35 long-nosed potoroos (Potorous tridactylus) were captured in state forests in South Eastern New South Wales for translocation to Booderee National Park, Jervis Bay Territory, Australia. Animals were anesthetized for physical examination and collection of samples to assess general health and screen for select diseases identified during a disease risk assessment. Morphologic, hematologic, and biochemical parameters were determined, and parasites were identified where possible. Trypanosoma gilletti, Trypanosoma vegrandis, and novel genotypes most similar to a Trypanosoma wallaby-derived isolate (ABF) were identified from blood samples by PCR; the first time Trypanosoma has been described in this species. Also reported is the first confirmation of the Australian paralysis tick, Ixodes holocyclus, from the long-nosed potoroo. Surveillance showed that Cryptococcus sp. may form part of the normal nasal flora for long-nosed potoroo. Salmonella enterica serotype Dublin and Salmonella enterica subsp. enterica was identified from rectal swabs of otherwise healthy animals. The data provide baseline health and disease parameters for this newly established population and the source population and will inform future translocation and conservation management activities. These data expand current knowledge on aspects of the biology and microbiology of the long-nosed potoroo, both locally and nationally.


Assuntos
Macropodidae , Parques Recreativos , Animais , Austrália/epidemiologia , Potoroidae , Salmonella
5.
Mol Ecol ; 30(8): 1777-1790, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33590590

RESUMO

While the effects of climate (long-term, prevailing weather) on species abundance, range and genetic diversity have been widely studied, short-term, localized variations in atmospheric conditions (i.e., weather) can also rapidly alter species' geographical ranges and population sizes, but little is known about how they affect genetic diversity. We investigated the relationship between weather and range-wide genetic diversity in a marsupial, Bettongia gaimardi, using dynamic species distribution models (SDMs). Genetic diversity was lower in parts of the range where the weather-based SDM predicted high variability in probability of B. gaimardi occurrence during 1950-2009. This is probably an effect of lower population sizes and extinction-recolonization cycles in places with highly variable weather. Spatial variation in genetic diversity was also better predicted by mean probabilities of B. gaimardi occurrence from weather- than climate-based SDMs. Our results illustrate the importance of weather in driving population dynamics and species distributions on decadal timescales and thereby in affecting genetic diversity. Modelling the links between changing weather patterns, species distributions and genetic diversity will allow researchers to better forecast biological impacts of climate change.


Assuntos
Mudança Climática , Tempo (Meteorologia) , Animais , Ecossistema , Variação Genética , Dinâmica Populacional , Potoroidae
6.
Biochim Biophys Acta Biomembr ; 1863(2): 183511, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33245892

RESUMO

Some anesthetics bind and potentiate γ-aminobutyric-acid-type receptors, but no universal mechanism for general anesthesia is known. Furthermore, often encountered complications such as anesthesia induced amnesia are not understood. General anesthetics are hydrophobic molecules easily dissolving into lipid bilayers. Recently, it was shown that general anesthetics perturb phase separation in vesicles extracted from fixed cells. Unclear is whether under physiological conditions general anesthetics induce perturbation of the lipid bilayer, and whether this contributes to the transient loss of consciousness or anesthesia side effects. Here we show that propofol perturbs lipid nanodomains in the outer and inner leaflet of the plasma membrane in intact cells, affecting membrane nanodomains in a concentration dependent manner: 1 µM to 5 µM propofol destabilize nanodomains; however, propofol concentrations higher than 5 µM stabilize nanodomains with time. Stabilization occurs only at physiological temperature and in intact cells. This process requires ARP2/3 mediated actin nucleation and Myosin II activity. The rate of nanodomain stabilization is potentiated by GABAA receptor activity. Our results show that active nanodomain homeostasis counteracts the initial disruption causing large changes in cortical actin. SIGNIFICANCE STATEMENT: General anesthesia is a routine medical procedure with few complications, yet a small number of patients experience side-effects that persist for weeks and months. Very young children are at risk for effects on brain development. Elderly patients often exhibit subsequent amnesia. Here, we show that the general anesthetic propofol perturbs the ultrastructure of the lipid bilayer of the cell membrane in intact cells. Initially propofol destabilized lipid nanodomains. However, with increasing incubation time and propofol concentration, the effect is reversed and nanodomains are further stabilized. We show that this stabilization is caused by the activation of the actin cortex under the membrane. These perturbations of membrane bilayer and cortical actin may explain how propofol affects neuronal plasticity at synapses.


Assuntos
Anestesia , Anestésicos Intravenosos/farmacologia , Homeostase/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Propofol/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Temperatura Alta , Masculino , Potoroidae
7.
Science ; 370(6519): 925, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33214273
8.
Zootaxa ; 4851(2): zootaxa.4851.2.3, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-33056727

RESUMO

A phylogenetic analysis of the genera of the strongyloid sub-family Cloacininae from macropodoid marsupials in Australasia was undertaken based on morphological characteristics and analysis of concatenated sequences (ITS+) of the first (ITS-1) and second (ITS-2) internal transcribed spacers of nuclear ribosomal DNA. Neither approach provided a robust phylogeny, but similarities between the two methods in terms of generic groupings suggested that substantial revision is needed of the current phenetic classification, with some of the key morphological characteristics currently used to define genera and tribes proving to be homoplasious.


Assuntos
Macropodidae , Nematoides , Animais , Filogenia , Potoroidae , Ratos , Strongyloidea
9.
PLoS One ; 15(4): e0227849, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343690

RESUMO

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.


Assuntos
Quebras de DNA/efeitos da radiação , Reparo do DNA , DNA/biossíntese , Fase G1/genética , Mitose/genética , Animais , Linhagem Celular , DNA/genética , DNA/efeitos da radiação , Fase G1/efeitos da radiação , Humanos , Raios Infravermelhos/efeitos adversos , Lasers/efeitos adversos , Mitose/efeitos da radiação , Potoroidae
10.
Genes (Basel) ; 10(11)2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661830

RESUMO

Small and isolated populations are subject to the loss of genetic variation as a consequence of inbreeding and genetic drift, which in turn, can affect the fitness and long-term viability of populations. Translocations can be used as an effective conservation tool to combat this loss of genetic diversity through establishing new populations of threatened species, and to increase total population size. Releasing animals from multiple genetically diverged sources is one method to optimize genetic diversity in translocated populations. However, admixture as a conservation tool is rarely utilized due to the risks of outbreeding depression. Using high-resolution genomic markers through double-digest restriction site-associated sequencing (ddRAD-seq) and life history data collected over nine years of monitoring, this study investigates the genetic and fitness consequences of admixing two genetically-distinct subspecies of Bettongia lesueur in a conservation translocation. Using single nucleotide polymorphisms (SNPs) identified from 215 individuals from multiple generations, we found an almost 2-fold increase in genetic diversity in the admixed translocation population compared to the founder populations, and this was maintained over time. Furthermore, hybrid class did not significantly impact on survivorship or the recruitment rate and therefore we found no indication of outbreeding depression. This study demonstrates the beneficial application of mixing multiple source populations in the conservation of threatened species for minimizing inbreeding and enhancing adaptive potential and overall fitness.


Assuntos
Genoma , Hibridização Genética , Polimorfismo de Nucleotídeo Único , Potoroidae/genética , Animais , Espécies em Perigo de Extinção , Aptidão Genética
11.
Genes (Basel) ; 10(9)2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546973

RESUMO

The use of multiple source populations provides a way to maximise genetic variation and reduce the impacts of inbreeding depression in newly established translocated populations. However, there is a risk that individuals from different source populations will not interbreed, leading to population structure and smaller effective population sizes than expected. Here, we investigate the genetic consequences of mixing two isolated, morphologically distinct island populations of boodies (Bettongia lesueur) in a translocation to mainland Australia over three generations. Using 18 microsatellite loci and the mitochondrial D-loop region, we monitored the released animals and their offspring between 2010 and 2013. Despite high levels of divergence between the two source populations (FST = 0.42 and ϕST = 0.72), there was clear evidence of interbreeding between animals from different populations. However, interbreeding was non-random, with a significant bias towards crosses between the genetically smaller-sized Barrow Island males and the larger-sized Dorre Island females. This pattern of introgression was opposite to the expectation that male-male competition or female mate choice would favour larger males. This study shows how mixing diverged populations can bolster genetic variation in newly established mammal populations, but the ultimate outcome can be difficult to predict, highlighting the need for continued genetic monitoring to assess the long-term impacts of admixture.


Assuntos
Introgressão Genética , Potoroidae/genética , Translocação Genética , Animais , Ecossistema , Feminino , Genótipo , Masculino
12.
Parasit Vectors ; 12(1): 126, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898141

RESUMO

BACKGROUND: Understanding how fauna translocation and antiparasitic drug treatment impact parasite community structure within a host is vital for optimising translocation outcomes. Trypanosoma spp. and piroplasms (Babesia and Theileria spp.) are known to infect Australian marsupials, including the woylie (Bettongia penicillata). However relatively little is known about these haemoparasites, or how they respond to management practices such as translocation. We monitored haemoparasites infecting woylies for up to 12 months during two fauna translocations to supplement existing woylie populations in three different sites (Dryandra, Walcott and Warrup East) within south-western Australia between 2014 and 2016, with the aim of investigating (i) how haemoparasite prevalence, Trypanosoma spp. richness and Trypanosoma spp. community composition varied over time and between different sites following translocation; and (ii) whether ivermectin treatment indirectly impacts haemoparasite prevalence. Using molecular methods, 1211 blood samples were screened for the presence of trypanosomes, and a subset of these samples (n = 264) were also tested for piroplasms. RESULTS: Trypanosomes and piroplasms were identified in 55% and 94% of blood samples, respectively. We identified five Trypanosoma species, two Theileria species, a single species of Babesia and a novel Bodo species. Trypanosoma spp. richness and the prevalence of haemoparasite co-infection increased after translocation. Prior to translocation, Trypanosoma spp. community composition differed significantly between translocated and resident woylies within Walcott and Warrup East, but not Dryandra. Six months later, there was a significant difference between translocated and resident woylies within Dryandra, but not Walcott or Warrup East. The response of haemoparasites to translocation was highly site-specific, with predominant changes to the haemoparasite community in translocated woylies occurring within the first few months following translocation. Ivermectin treatment had no significant effect on haemoparasite prevalence. CONCLUSIONS: This study contributes to our understanding of haemoparasite dynamics in woylies following translocation. The highly site-specific and rapid response of haemoparasites to translocation highlights the need to better understand what drives these effects. Given that haemoparasite prevalence and composition of translocated and resident animals changed significantly following translocation, we propose that parasite monitoring should form an essential component of translocation protocols, and such protocols should endeavour to monitor translocated hosts and cohabiting species.


Assuntos
Potoroidae/parasitologia , Tripanossomíase/veterinária , Animais , Antiprotozoários/administração & dosagem , Austrália/epidemiologia , Babesia , Babesiose/sangue , Babesiose/complicações , Babesiose/epidemiologia , Coinfecção/veterinária , Feminino , Ivermectina/administração & dosagem , Masculino , Filogeografia , Prevalência , Theileria , Theileriose/sangue , Theileriose/complicações , Theileriose/epidemiologia , Trypanosoma , Tripanossomíase/complicações , Tripanossomíase/epidemiologia , Tripanossomíase/parasitologia
13.
Anat Rec (Hoboken) ; 301(12): 2086-2094, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30312007

RESUMO

Salmonella enterica serovar Typhimurium (S. Typhimurium), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) commandeer the actin cytoskeleton of their host cells as a crucial step in their infectious processes. These pathogens depend on the injection of their own effectors directly into target host cells in order to usurp cellular signaling pathways that lead to morphological actin rearrangements in those cells. Here we show that the PPIase Cyclophilin A (CypA) is a novel component of S. Typhimurium-induced membrane ruffles and functions to restrict bacterial invasion levels, as in cells depleted of CypA, bacterial loads increase. We also demonstrate that CypA requires the EPEC effector Tir as well as pedestal formation for its recruitment to bacterial attachment sites and that its presence at pedestals also holds during EHEC infections. Finally, we demonstrate that CypA is found at lamellipodia; actin-rich structures at the leading edge of motile cells. Our findings further establish CypA as a component of dynamic actin-rich structures formed during bacterial infections and within cells in general. Anat Rec, 301:2086-2094, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Ciclofilina A/metabolismo , Escherichia coli/metabolismo , Salmonella/metabolismo , Citoesqueleto de Actina/química , Actinas/análise , Animais , Ciclofilina A/análise , Células HeLa , Humanos , Camundongos , Potoroidae
14.
Mol Ecol ; 27(23): 4960-4971, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30375068

RESUMO

Organisms that are highly connected in food webs often perform unique and vital functions within ecosystems. Understanding the unique ecological roles played by highly connected organisms and the consequences of their loss requires a comprehensive understanding of the functional redundancy amongst organisms. One important, yet poorly understood, food web is that between truffle-forming ectomycorrhizal fungi and their mammalian consumers and dispersers. Mammalian fungal specialists rely on fungi as a food source, and they consume and disperse a higher diversity and abundance of fungi than do mycophagous mammals with generalist diets. Therefore, we hypothesize that mammalian fungal specialists are functionally distinct because they disperse a set of fungal taxa not fully nested within the set consumed by the combined generalist mammalian community (i.e., functional redundancy of fungal dispersal is limited). Using high-throughput sequencing, we compared the fungal composition of 93 scats from the endangered fungal specialist northern bettong (Bettongia tropica) and 120 scats from nine co-occurring generalist mammal species across three sites and three seasons. Compared with other generalist mammals, B. tropica consumed a more diverse fungal diet with more unique taxa. This aligns with our hypothesis that B. tropica performs a unique dispersal function for ectomycorrhizal truffle fungi. Additionally, modelling of mammalian extinctions predicted rapid loss of food web connections which could result in loss of gene flow for truffle taxa. Our results suggest that this system is sensitive to the extinction of highly connected specialist species like B. tropica and their loss could have consequences for ectomycorrhizal truffle fungal diversity. This suggests that the conservation of fungal specialists is imperative to maintaining ectomycorrhizal fungal diversity and healthy plant-mycorrhizal relationships.


Assuntos
Cadeia Alimentar , Micorrizas/classificação , Potoroidae/microbiologia , Animais , Biodiversidade , Espécies em Perigo de Extinção , Mamíferos , Queensland , Microbiologia do Solo
15.
J Cell Biol ; 217(8): 2691-2708, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29794031

RESUMO

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.


Assuntos
Microtúbulos/metabolismo , Tubulina (Proteína)/fisiologia , Animais , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Linhagem Celular , Chlamydomonas/metabolismo , Chlamydomonas/ultraestrutura , Tomografia com Microscopia Eletrônica , Guanosina Trifosfato/metabolismo , Microtúbulos/química , Microtúbulos/ultraestrutura , Potoroidae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura , Tubulina (Proteína)/metabolismo
16.
Exp Cell Res ; 369(1): 139-146, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29778753

RESUMO

The host actin cytoskeleton is utilized by an assortment of pathogenic bacteria to colonize and cause disease in their hosts. Two prominently studied actin-hijacking bacteria are enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes. EPEC form actin-rich pedestals atop its host cells to move across the intestinal epithelia, while Listeria monocytogenes generate branched actin networks arranged as actin clouds around the bacteria and as comet tails for propulsion within and amongst their host cells. Previous mass spectrometry analysis revealed that a member of the calponin family of actin-bundling proteins, transgelin/SM22 was enriched in EPEC pedestals. To validate that finding and examine the role of SM22 during infections, we initially immunolocalized SM22 in EPEC and L. monocytogenes infected cells, used siRNA to deplete SM22 and EGFP-SM22 to overexpress SM22, then quantified the alterations to the bacterially generated actin structures. SM22 concentrated at all bacterially-generated actin structures. Depletion of SM22 resulted in fewer pedestals and comet tails and caused comet tails to shorten. The decrease in comet tail abundance caused a proportional increase in actin clouds whereas overexpression of SM22 reversed the actin cloud to comet tail proportions and increased comet tail length, while not influencing EPEC pedestal abundance. Thus, we demonstrate that SM22 plays a role in regulating the transitions and morphological appearance of bacterially generated actin-rich structures during infections.


Assuntos
Actinas/metabolismo , Infecções Bacterianas/metabolismo , Estruturas Celulares/metabolismo , Estruturas Celulares/microbiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas Musculares/fisiologia , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/patologia , Células CACO-2 , Células Cultivadas , Estruturas Celulares/patologia , Escherichia coli Enteropatogênica , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Células HeLa , Humanos , Listeria monocytogenes , Listeriose/genética , Listeriose/metabolismo , Potoroidae
17.
Nat Commun ; 9(1): 1688, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703977

RESUMO

Cell protrusion is morphodynamically heterogeneous at the subcellular level. However, the mechanism of cell protrusion has been understood based on the ensemble average of actin regulator dynamics. Here, we establish a computational framework called HACKS (deconvolution of heterogeneous activity in coordination of cytoskeleton at the subcellular level) to deconvolve the subcellular heterogeneity of lamellipodial protrusion from live cell imaging. HACKS identifies distinct subcellular protrusion phenotypes based on machine-learning algorithms and reveals their underlying actin regulator dynamics at the leading edge. Using our method, we discover "accelerating protrusion", which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. We validate our finding by pharmacological perturbations and further identify the fine regulation of Arp2/3 and VASP recruitment associated with accelerating protrusion. Our study suggests HACKS can identify specific subcellular protrusion phenotypes susceptible to pharmacological perturbation and reveal how actin regulator dynamics are changed by the perturbation.


Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Aprendizado de Máquina , Modelos Biológicos , Pseudópodes/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Análise por Conglomerados , Humanos , Indóis/farmacologia , Microscopia Intravital , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Potoroidae , Software
18.
J Cell Biol ; 217(1): 163-177, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29187526

RESUMO

Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.


Assuntos
Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Linhagem Celular Tumoral , Centrômero/metabolismo , Proteínas do Citoesqueleto , Células HeLa , Humanos , Metáfase/fisiologia , Fosforilação , Potoroidae , Ligação Proteica/fisiologia , Fuso Acromático/metabolismo
19.
J Am Chem Soc ; 139(32): 10944-10947, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28774176

RESUMO

By recording the full fluorescence spectra and super-resolved positions of ∼106 individual polarity-sensing solvatochromic molecules, we reveal compositional heterogeneity in the membranes of live mammalian cells with single-molecule sensitivity and ∼30 nm spatial resolution. This allowed us to unveil distinct polarity characteristics of the plasma membrane and the membranes of nanoscale intracellular organelles, a result we found to be due to differences in cholesterol levels. Within the plasma membrane, we observed the formation of low-polarity, raft-like nanodomains upon cholesterol addition or cholera-toxin treatment, but found this nanoscale phase separation absent in native cells. The ultimate sensitivity achieved through examining the spectra of individual molecules thus opens the door to functional interrogations of intracellular physicochemical parameters at the nanoscale.


Assuntos
Membrana Celular/ultraestrutura , Colesterol/análise , Retículo Endoplasmático/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Espectrometria de Fluorescência/métodos , Animais , Células COS , Linhagem Celular , Membrana Celular/química , Sobrevivência Celular , Chlorocebus aethiops , Retículo Endoplasmático/química , Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Membranas Mitocondriais/química , Oxazinas/análise , Potoroidae
20.
Curr Biol ; 27(14): 2112-2122.e5, 2017 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-28690110

RESUMO

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore fibers (k-fibers) to bear this load. The mammalian spindle bears the load of chromosome movement far from poles, but we do not know where and how-physically and molecularly-this load distributes across the spindle. In part, this is because probing spindle mechanics in live cells is difficult. Yet answering this question is key to understanding how the spindle generates and responds to force and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time and dissect local anchorage mechanics and mechanism. To do so, we laser-ablate single k-fibers at different spindle locations and in different molecular backgrounds and quantify the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 µm from kinetochores, scaling with k-fiber length, and laterally within ∼2 µm of k-fiber sides, without detectable load sharing between neighboring k-fibers. A phenomenological model suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, whereas Eg5 and PRC1 are not detectably required, suggesting specialization in mechanical function. Together, the data and model suggest that NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well suited to support robust chromosome segregation.


Assuntos
Cinetocoros/fisiologia , Fuso Acromático/fisiologia , Animais , Linhagem Celular , Cinética , Potoroidae
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...