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1.
J Exp Med ; 216(5): 1050-1060, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-30914438

RESUMO

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

4.
Am J Hum Genet ; 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

5.
Open Biol ; 8(10)2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30282661

RESUMO

Repair of wounds to individual cells is crucial for organisms to survive daily physiological or environmental stresses, as well as pathogen assaults, which disrupt the plasma membrane. Sensing wounds, resealing membranes, closing wounds and remodelling plasma membrane/cortical cytoskeleton are four major steps that are essential to return cells to their pre-wounded states. This process relies on dynamic changes of the membrane/cytoskeleton that are indispensable for carrying out the repairs within tens of minutes. Studies from different cell wound repair models over the last two decades have revealed that the molecular mechanisms of single cell wound repair are very diverse and dependent on wound type, size, and/or species. Interestingly, different repair models have been shown to use similar proteins to achieve the same end result, albeit sometimes by distinctive mechanisms. Recent studies using cutting edge microscopy and molecular techniques are shedding new light on the molecular mechanisms during cellular wound repair. Here, we describe what is currently known about the mechanisms underlying this repair process. In addition, we discuss how the study of cellular wound repair-a powerful and inducible model-can contribute to our understanding of other fundamental biological processes such as cytokinesis, cell migration, cancer metastasis and human diseases.

7.
J Cell Sci ; 131(8)2018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29549166

RESUMO

WASH, a Wiskott-Aldrich syndrome (WAS) family protein, has many cell and developmental roles related to its function as a branched actin nucleation factor. Similar to mammalian WASHC1, which is embryonic lethal, Drosophila Wash was found to be essential for oogenesis and larval development. Recently, however, Drosophila wash was reported to be homozygous viable. Here, we verify that the original wash null allele harbors an unrelated lethal background mutation; however, this unrelated lethal mutation does not contribute to any Wash oogenesis phenotypes. Significantly, we find that: (1) the homozygous wash null allele retains partial lethality, leading to non-Mendelian inheritance; (2) the allele's functions are subject to its specific genetic background; and (3) the homozygous stock rapidly accumulates modifications that allow it to become robust. Together, these results suggest that Wash plays an important role in oogenesis via the WASH regulatory complex. Finally, we show that another WAS family protein, SCAR/WAVE, plays a similar role in oogenesis and that it is upregulated as one of the modifications that allows the wash allele to survive in the homozygous state.

8.
J Cell Biol ; 216(12): 3959-3969, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-28923977

RESUMO

Like tissues, single cells are subjected to continual stresses and damage. As such, cells have a robust wound repair mechanism comprised of dynamic membrane resealing and cortical cytoskeletal remodeling. One group of proteins, the Rho family of small guanosine triphosphatases (GTPases), is critical for this actin and myosin cytoskeletal response in which they form distinct dynamic spatial and temporal patterns/arrays surrounding the wound. A key mechanistic question, then, is how these GTPase arrays are formed. Here, we show that in the Drosophila melanogaster cell wound repair model Rho GTPase arrays form in response to prepatterning by Rho guanine nucleotide exchange factors (RhoGEFs), a family of proteins involved in the activation of small GTPases. Furthermore, we show that Annexin B9, a member of a class of proteins associated with the membrane resealing, is involved in an early, Rho family-independent, actin stabilization that is integral to the formation of one RhoGEF array. Thus, Annexin proteins may link membrane resealing to cytoskeletal remodeling processes in single cell wound repair.


Assuntos
Anexinas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas rho de Ligação ao GTP/genética , Actinas/genética , Actinas/metabolismo , Animais , Anexinas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Embrião não Mamífero , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Modelos Biológicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Cicatrização/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo
9.
Nucleus ; 6(5): 349-59, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26305109

RESUMO

Actin and proteins that regulate its dynamics or interactions have well-established roles in the cytoplasm where they function as key components of the cytoskeleton to control diverse processes, including cellular infrastructure, cellular motility, cell signaling, and vesicle transport. Recent work has also uncovered roles for actin and its regulatory proteins in the nucleus, primarily in mechanisms governing gene expression. The Wiskott Aldrich Syndrome (WAS) family of proteins, comprising the WASP/N-WASP, SCAR/WAVE, WHAMM/JMY/WHAMY, and WASH subfamilies, function in the cytoplasm where they activate the Arp2/3 complex to form branched actin filaments. WAS proteins are present in the nucleus and have been implicated as transcriptional regulators. We found that Drosophila Wash, in addition to transcriptional effects, is involved in global nuclear architecture. Here we summarize the regulation and function of nuclear WAS proteins, and highlight how our work with Wash expands the possibilities for the functions of these proteins in the nucleus.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Laminas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Feminino , Masculino
10.
Small GTPases ; 6(1): 1-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25862160

RESUMO

Repair of wounds to single cells involves dynamic membrane and cytoskeletal rearrangements necessary to seal the wound and repair the underlying cytoskeleton cortex. One group of proteins essential to the cortical remodeling is the Rho family of small GTPases. Recently we showed that the founding members of this GTPases family, Rho, Rac, and Cdc42, are all essential for normal single cell wound repair and accumulate at the wound periphery in distinct temporal/spatial patterns in the Drosophila cell wound model. In addition, these proteins communicate with one another and with the cytoskeleton to regulate their distribution in response to wounds. Unexpectedly, we found evidence for context specific Rho GTPase binding to downstream targets or "effectors" which cannot be explained solely by means of local GTPase activation. Here we discuss these observations in relation to similar studies in single cell wound repair in the Xenopus oocyte, and highlight how these cell wound models serve as powerful tools to understand both cell wound repair and Rho GTPase biology.


Assuntos
Cicatrização , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Citoesqueleto/enzimologia , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Drosophila , Humanos , Família Multigênica , Xenopus , Proteínas rho de Ligação ao GTP/genética
11.
Small GTPases ; 6(1): 28-35, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25862164

RESUMO

Epithelial repair in the Drosophila embryo is achieved through 2 dynamic cytoskeletal machineries: a contractile actomyosin cable and actin-based cellular protrusions. Rho family small GTPases (Rho, Rac, and Cdc42) are cytoskeletal regulators that control both of these wound repair mechanisms. Cdc42 is necessary for cellular protrusions and, when absent, wounds are slow to repair and never completely close. Rac proteins accumulate at specific regions in the wound leading edge cells and Rac-deficient embryos exhibit slower repair kinetics. Mutants for both Rho1 and its effector Rok impair the ability of wounds to close by disrupting the leading-edge actin cable. Our studies highlight the importance of these proteins in wound repair and identify a downstream effector of Rho1 signaling in this process.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , Epitélio/enzimologia , Cicatrização , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Drosophila/embriologia , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Epitélio/fisiologia , Feminino , Masculino , Família Multigênica , Proteínas rho de Ligação ao GTP/genética
12.
Curr Biol ; 25(6): 804-810, 2015 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-25754639

RESUMO

The cytoplasmic functions of Wiskott-Aldrich syndrome family (WAS) proteins are well established and include roles in cytoskeleton reorganization and membrane-cytoskeletal interactions important for membrane/vesicle trafficking, morphogenesis, immune response, and signal transduction. Misregulation of these proteins is associated with immune deficiency and metastasis [1-4]. Cytoplasmic WAS proteins act as effectors of Rho family GTPases and polymerize branched actin through the Arp2/3 complex [1, 5]. Previously, we identified Drosophila washout (wash) as a new member of the WAS family with essential cytoplasmic roles in early development [6, 7]. Studies in mammalian cells and Dictyostelium suggest that WASH functions primarily in a multiprotein complex that regulates endosome shape and trafficking in an Arp2/3-dependent manner [8-11]. However, roles for classically cytoplasmic proteins in the nucleus are beginning to emerge, in particular, as participants in the regulation of gene expression [12, 13]. Here, we show that Drosophila Wash is present in the nucleus, where it plays a key role in global nuclear organization. wash mutant and knockdown nuclei disrupt subnuclear structures/organelles and exhibit the abnormal wrinkled morphology reminiscent of those observed in diverse laminopathies [14-16]. We find that nuclear Wash interacts with B-type Lamin (Lamin Dm0), and, like Lamin, Wash associates with constitutive heterochromatin. Wash knockdown increases chromatin accessibility of repressive compartments and results in a global redistribution of repressive histone modifications. Thus, our results reveal a novel role for Wash in modulating nucleus morphology and in the organization of both chromatin and non-chromatin nuclear sub-structures.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Laminas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Feminino , Técnicas de Silenciamento de Genes , Genes de Insetos , Heterocromatina/genética , Heterocromatina/metabolismo , Laminas/genética , Masculino , Mutação , Proteínas de Transporte Vesicular/genética
13.
Mol Biol Cell ; 26(9): 1665-74, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25739458

RESUMO

Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non-immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott-Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex-independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations.


Assuntos
Movimento Celular , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Hemócitos/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Drosophila melanogaster/citologia , Hemócitos/ultraestrutura , Ligação Proteica
14.
Curr Biol ; 24(2): 144-55, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24388847

RESUMO

BACKGROUND: Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. Here we describe how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. RESULTS: We find that Rho, Rac, and Cdc42 rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. We find Rho1 GTPase uses several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. CONCLUSIONS: Our results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs.


Assuntos
Citoesqueleto/enzimologia , Drosophila melanogaster/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Dactinomicina/química , Drosophila melanogaster/citologia , Ativação Enzimática
15.
J Cell Sci ; 125(Pt 24): 5984-97, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23038780

RESUMO

The repair of injured tissue must occur rapidly to prevent microbial invasion and maintain tissue integrity. Epithelial tissues in particular, which serve as a barrier against the external environment, must repair efficiently in order to restore their primary function. Here we analyze the effect of different parameters on the epithelial wound repair process in the late stage Drosophila embryo using in vivo wound assays, expression of cytoskeleton and membrane markers, and mutant analysis. We define four distinct phases in the repair process, expansion, coalescence, contraction and closure, and describe the molecular dynamics of each phase. Specifically, we find that myosin, E-cadherin, Echinoid, the plasma membrane, microtubules and the Cdc42 small GTPase respond dynamically during wound repair. We demonstrate that perturbations of each of these components result in specific impairments to the wound healing process. Our results show that embryonic epithelial wound repair is mediated by two simultaneously acting mechanisms: crawling driven by cellular protrusions and actomyosin ring contraction along the leading edge of the wound.


Assuntos
Actomiosina/metabolismo , Drosophila/metabolismo , Animais , Drosophila/citologia , Drosophila/embriologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo
16.
Cell Mol Life Sci ; 69(15): 2469-83, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22349211

RESUMO

Wound repair on the cellular and multicellular levels is essential to the survival of complex organisms. In order to avoid further damage, prevent infection, and restore normal function, cells and tissues must rapidly seal and remodel the wounded area. The cytoskeleton is an important component of wound repair in that it is needed for actomyosin contraction, recruitment of repair machineries, and cell migration. Recent use of model systems and high-resolution microscopy has provided new insight into molecular aspects of the cytoskeletal response during wound repair. Here we discuss the role of the cytoskeleton in single-cell, embryonic, and adult repair, as well as the striking resemblance of these processes to normal developmental events and many diseases.


Assuntos
Citoesqueleto/fisiologia , Cicatrização/fisiologia , Actomiosina/fisiologia , Animais , Caderinas/fisiologia , Sinalização do Cálcio/fisiologia , Membrana Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Embrião de Mamíferos/fisiopatologia , Embrião não Mamífero/lesões , Embrião não Mamífero/fisiopatologia , Humanos , Modelos Biológicos , Morfogênese/fisiologia , Lesões Pré-Natais/fisiopatologia
17.
Bioarchitecture ; 1(3): 114-121, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21922041

RESUMO

Cell wounding is a common event in the life of many cell types, and the capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Cell wounding is most frequent in tissues exposed to high levels of stress. Survival of such plasma membrane disruptions requires rapid resealing to prevent the loss of cytosolic components, to block Ca(2+) influx and to avoid cell death. In addition to patching the torn membrane, plasma membrane and cortical cytoskeleton remodeling are required to restore cell function. Although a general understanding of the cell wound repair process is in place, the underlying mechanisms of each step of this response are not yet known. We have developed a model to study single cell wound repair using the early Drosophila embryo. Our system combines genetics and live imaging tools, allowing us to dissect in vivo the dynamics of the single cell wound response. We have shown that cell wound repair in Drosophila requires the coordinated activities of plasma membrane and cytoskeleton components. Furthermore, we identified an unexpected role for E-cadherin as a link between the contractile actomyosin ring and the newly formed plasma membrane plug.

18.
J Cell Biol ; 193(3): 455-64, 2011 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-21518790

RESUMO

When single cells or tissues are injured, the wound must be repaired quickly in order to prevent cell death, loss of tissue integrity, and invasion by microorganisms. We describe Drosophila as a genetically tractable model to dissect the mechanisms of single-cell wound repair. By analyzing the expression and the effects of perturbations of actin, myosin, microtubules, E-cadherin, and the plasma membrane, we define three distinct phases in the repair process-expansion, contraction, and closure-and identify specific components required during each phase. Specifically, plasma membrane mobilization and assembly of a contractile actomyosin ring are required for this process. In addition, E-cadherin accumulates at the wound edge, and wound expansion is excessive in E-cadherin mutants, suggesting a role for E-cadherin in anchoring the actomyosin ring to the plasma membrane. Our results show that single-cell wound repair requires specific spatial and temporal cytoskeleton responses with distinct components and mechanisms required at different stages of the process.


Assuntos
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Actomiosina/química , Alelos , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Cruzamentos Genéticos , Microtúbulos/metabolismo , Mutação , Miosinas/metabolismo , Cicatrização
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