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1.
J Cell Sci ; 137(6)2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38345097

RESUMO

Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Junções Intercelulares/metabolismo , Lipoilação , Proteolipídeos/metabolismo
2.
Development ; 150(2)2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-36628974

RESUMO

Src kinases are important regulators of cell adhesion. Here, we have explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. We find that, during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, we show that Src42A acts in concert with Abl kinase, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Junções Aderentes/metabolismo , Caderinas/genética , Caderinas/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Junções Intercelulares/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
4.
J Cell Sci ; 135(3)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35019140

RESUMO

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We define two distinct EV subpopulations, one of which contains the Munc13-4 (also known as UNC13D) homolog Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3 (also known as Dnd in Drosophila), which is also required for the formation of Stac-positive multivesicular bodies (MVBs), suggesting that Stac-positive EVs derive from fusion of Stac-positive MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-positive MVB formation and tube fusion. We propose that Stac-positive MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35 and Stac. This article has an associated First Person interview with the first author of the paper.


Assuntos
Vesículas Extracelulares , Proteínas Monoméricas de Ligação ao GTP , Traqueia/crescimento & desenvolvimento , Animais , Drosophila , Morfogênese , Corpos Multivesiculares
5.
Cell Mol Life Sci ; 79(5): 248, 2022 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-35437696

RESUMO

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitose , Mamíferos/metabolismo , Fosfatidilinositóis/metabolismo
6.
PLoS Biol ; 16(4): e2004718, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29702642

RESUMO

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Drosophila melanogaster/ultraestrutura , Miofibrilas/ultraestrutura , Pupa/ultraestrutura , Sarcômeros/ultraestrutura , Citoesqueleto de Actina/metabolismo , Actinas/ultraestrutura , Animais , Fenômenos Biomecânicos , Conectina/metabolismo , Conectina/ultraestrutura , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Voo Animal/fisiologia , Microscopia de Polarização/métodos , Miofibrilas/metabolismo , Miosinas/metabolismo , Miosinas/ultraestrutura , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Sarcômeros/metabolismo
7.
Development ; 144(4): 657-663, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087625

RESUMO

Morphogenesis requires the dynamic regulation of gene expression, including transcription, mRNA maturation and translation. Dysfunction of the general mRNA splicing machinery can cause surprisingly specific cellular phenotypes, but the basis for these effects is not clear. Here, we show that the Drosophila faint sausage (fas) locus, which is implicated in epithelial morphogenesis and has previously been reported to encode a secreted immunoglobulin domain protein, in fact encodes a subunit of the spliceosome-activating Prp19 complex, which is essential for efficient pre-mRNA splicing. Loss of zygotic fas function globally impairs the efficiency of splicing, and is associated with widespread retention of introns in mRNAs and dramatic changes in gene expression. Surprisingly, despite these general effects, zygotic fas mutants show specific defects in tracheal cell migration during mid-embryogenesis when maternally supplied splicing factors have declined. We propose that tracheal branching, which relies on dynamic changes in gene expression, is particularly sensitive for efficient spliceosome function. Our results reveal an entry point to study requirements of the splicing machinery during organogenesis and provide a better understanding of disease phenotypes associated with mutations in general splicing factors.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Neuropeptídeos/metabolismo , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Traqueia/embriologia , Alelos , Animais , Movimento Celular , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imunoglobulinas/metabolismo , Íntrons , Masculino , Morfogênese , Mutação , Precursores de RNA/genética , Fatores de Processamento de RNA/fisiologia , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Traqueia/metabolismo
8.
PLoS Genet ; 12(5): e1006073, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27223464

RESUMO

Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , MicroRNAs/biossíntese , Prolil Hidroxilases/genética , Transcrição Gênica , Animais , Hipóxia Celular/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/genética , Oxigênio/metabolismo , Prolil Hidroxilases/metabolismo
9.
Development ; 141(4): 899-908, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24496626

RESUMO

Occluding cell-cell junctions in epithelia form physical barriers that separate different membrane domains, restrict paracellular diffusion and prevent pathogens from spreading across tissues. In invertebrates, these functions are provided by septate junctions (SJs), the functional equivalent of vertebrate tight junctions. How the diverse functions of SJs are integrated and modulated in a multiprotein complex is not clear, and many SJ components are still unknown. Here we report the identification of Macroglobulin complement-related (Mcr), a member of the conserved α-2-macroglobulin (α2M) complement protein family, as a novel SJ-associated protein in Drosophila. Whereas α2M complement proteins are generally known as secreted factors that bind to surfaces of pathogens and target them for phagocytic uptake, Mcr represents an unusual α2M protein with a predicted transmembrane domain. We show that Mcr protein localizes to lateral membranes of epithelial cells, where its distribution overlaps with SJs. Several SJ components are required for the correct localization of Mcr. Conversely, Mcr is required in a cell-autonomous fashion for the correct membrane localization of SJ components, indicating that membrane-bound rather than secreted Mcr isoforms are involved in SJ formation. Finally, we show that loss of Mcr function leads to morphological, ultrastructural and epithelial barrier defects resembling mutants lacking SJ components. Our results, along with previous findings on the role of Mcr in phagocytosis, suggest that Mcr plays dual roles in epithelial barrier formation and innate immunity. Thus, Mcr represents a novel paradigm for investigating functional links between occluding junction formation and pathogen defense mechanisms.


Assuntos
Citocinas/fisiologia , Drosophila/genética , Células Epiteliais/fisiologia , Imunidade Inata/genética , Junções Intercelulares/fisiologia , Proteínas de Membrana/fisiologia , Serpinas/fisiologia , Animais , Drosophila/fisiologia , Proteínas de Drosophila , Imunofluorescência , Processamento de Imagem Assistida por Computador , Immunoblotting , Hibridização In Situ , Junções Intercelulares/genética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Oligonucleotídeos/genética
10.
Semin Cell Dev Biol ; 31: 82-90, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24662893

RESUMO

Organs like the vertebrate vascular system and the insect tracheal system develop from separate primordia that undergo fusion events to form interconnected tubular networks. Although the correct pattern of tubular connections (anastomoses) in these organs is crucial for their normal function, the cellular and molecular mechanisms that govern tube fusion are only beginning to be understood. The process of tube fusion involves tip cell specification, cell-cell recognition and contact formation, self-avoidance, changes in cell shape and topology, lumen formation, and luminal membrane fusion. Significant insights into the underlying cellular machinery have been provided by genetic studies of tracheal tube fusion in Drosophila. Here, we summarize these findings and we highlight similarities and differences between tube fusion processes in the Drosophila tracheae and in the vertebrate vascular system. We integrate the findings from studies in vivo with the important mechanistic insights that have been gained from the analysis of tubulogenesis in cultured cells to propose a mechanistic model of tube fusion, aspects of which are likely to apply to diverse organs and organisms.


Assuntos
Fusão de Membrana , Traqueia/metabolismo , Animais , Morfogênese , Traqueia/citologia , Traqueia/embriologia
11.
Development ; 140(6): 1240-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23444354

RESUMO

Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. Here, we show that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. We demonstrate that Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas do Olho/química , Proteínas do Olho/fisiologia , Sistema de Sinalização das MAP Quinases , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Traqueia/embriologia , Proteína Wnt1/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação para Baixo/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Sistema de Sinalização das MAP Quinases/genética , Morfogênese/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-ets/química , Proteínas Repressoras/genética , Transdução de Sinais/genética , Traqueia/citologia , Traqueia/metabolismo , Proteína Wnt1/antagonistas & inibidores
12.
Exp Cell Res ; 321(1): 64-70, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24075963

RESUMO

Tubular epithelia come in various shapes and sizes to accommodate the specific needs for transport, excretion and absorption in multicellular organisms. The intestinal tract, glandular organs and conduits for liquids and gases are all lined by a continuous layer of epithelial cells, which form the boundary of the luminal space. Defects in epithelial architecture and lumen dimensions will impair transport and can lead to serious organ malfunctions. Not surprisingly, multiple cellular and molecular mechanisms contribute to the shape of tubular epithelial structures. One intriguing aspect of epithelial organ formation is the highly coordinate behavior of individual cells as they mold the mature lumen. Here, we focus on recent findings, primarily from Drosophila, demonstrating that informative cues can emanate from the developing organ lumen in the form of solid luminal material. The luminal material is produced by the surrounding epithelium and helps to coordinate changes in shape and arrangement of the very same cells, resulting in correct lumen dimensions.


Assuntos
Drosophila/crescimento & desenvolvimento , Matriz Extracelular , Organogênese/fisiologia , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo
13.
J Cell Sci ; 125(Pt 5): 1318-28, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22349697

RESUMO

Protein trafficking through the secretory pathway plays a key role in epithelial organ development and function. The expansion of tracheal tubes in Drosophila depends on trafficking of coatomer protein complex I (COPI)-coated vesicles between the Golgi complex and the endoplasmic reticulum (ER). However, it is not clear how this pathway is regulated. Here we describe an essential function of the Sec7 domain guanine nucleotide exchange factor (GEF) gartenzwerg (garz) in epithelial tube morphogenesis and protein secretion. garz is essential for the recruitment of COPI components and for normal Golgi organization. A GFP-Garz fusion protein is distributed in the cytoplasm and accumulates at the cis-Golgi. Localization to the Golgi requires the C-terminal part of Garz. Conversely, blocking the GDP-GTP nucleotide exchange reaction leads to constitutive Golgi localization, suggesting that Garz cycles in a GEF-activity-dependent manner between cytoplasmic and Golgi-membrane-localized pools. The related human ARF-GEF protein GBF1 can substitute for garz function in Drosophila tracheal cells, indicating that the relevant functions of these proteins are conserved. We show that garz interacts genetically with the ARF1 homolog ARF79F and with the ARF1-GAP homolog Gap69C, thus placing garz in a regulatory circuit that controls COPI trafficking in Drosophila. Interestingly, overexpression of garz causes accumulation of secreted proteins in the ER, suggesting that excessive garz activity leads to increased retrograde trafficking. Thus, garz might regulate epithelial tube morphogenesis and secretion by controlling the rate of trafficking of COPI vesicles.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transporte Proteico/fisiologia , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Complexo I de Proteína do Envoltório/metabolismo , Proteína Coatomer/metabolismo , Retículo Endoplasmático , Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Traqueia/crescimento & desenvolvimento , Traqueia/metabolismo
14.
Dev Cell ; 58(8): 709-723.e7, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-37023749

RESUMO

Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but so far, tools for investigating the dynamics of membrane trafficking have been limited to cultured cells. Here, we present a system that enables acute manipulation and real-time visualization of membrane trafficking through the reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualizing and manipulating membrane trafficking in diverse cell types in vivo.


Assuntos
Drosophila , Complexo de Golgi , Animais , Transporte Proteico/fisiologia , Complexo de Golgi/metabolismo , Transporte Biológico , Exocitose
15.
Dev Cell ; 57(16): 2026-2040.e5, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35914525

RESUMO

Cell ablation is a key method in the research fields of developmental biology, tissue regeneration, and tissue homeostasis. Eliminating specific cell populations allows for characterizing interactions that control cell differentiation, death, behavior, and spatial organization of cells. Current methodologies for inducing cell death suffer from relatively slow kinetics, making them unsuitable for analyzing rapid events and following primary and immediate consequences of the ablation. To address this, we developed a cell-ablation system that is based on bacterial toxin/anti-toxin proteins and enables rapid and cell-autonomous elimination of specific cell types and organs in zebrafish embryos. A unique feature of this system is that it uses an anti-toxin, which allows for controlling the degree and timing of ablation and the resulting phenotypes. The transgenic zebrafish generated in this work represent a highly efficient tool for cell ablation, and this approach is applicable to other model organisms as demonstrated here for Drosophila.


Assuntos
Drosophila , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Morte Celular , Diferenciação Celular , Peixe-Zebra/genética
16.
J Cell Sci ; 122(Pt 20): 3759-71, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19789180

RESUMO

Polarity of many cell types is controlled by a protein complex consisting of Bazooka/PAR-3 (Baz), PAR-6 and atypical protein kinase C (aPKC). In Drosophila, the Baz-PAR-6-aPKC complex is required for the control of cell polarity in the follicular epithelium, in ectodermal epithelia and neuroblasts. aPKC is the main signaling component of this complex that functions by phosphorylating downstream targets, while the PDZ domain proteins Baz and PAR-6 control the subcellular localization and kinase activity of aPKC. We compared the mutant phenotypes of an aPKC null allele with those of four novel aPKC alleles harboring point mutations that abolish the kinase activity or the binding of aPKC to PAR-6. We show that these point alleles retain full functionality in the control of follicle cell polarity, but produce strong loss-of-function phenotypes in embryonic epithelia and neuroblasts. Our data, combined with molecular dynamics simulations, show that the kinase activity of aPKC and its ability to bind PAR-6 are only required for a subset of its functions during development, revealing tissue-specific differences in the way that aPKC controls cell polarity.


Assuntos
Drosophila melanogaster/enzimologia , Proteína Quinase C/metabolismo , Trifosfato de Adenosina/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Células Clonais , Sequência Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/enzimologia , Embrião não Mamífero/citologia , Embrião não Mamífero/enzimologia , Feminino , Células Germinativas/citologia , Células Germinativas/enzimologia , Homozigoto , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Neurônios/citologia , Neurônios/enzimologia , Oócitos/citologia , Oócitos/enzimologia , Dobramento de Proteína , Transporte Proteico
17.
Curr Top Dev Biol ; 143: 37-74, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33820625

RESUMO

Tubular networks, such as the vascular and respiratory systems, transport liquids and gases in multicellular organisms. The basic units of these organs are tubes formed by single or multiple cells enclosing a luminal cavity. The formation and maintenance of correctly sized and shaped lumina are fundamental steps in organogenesis and are essential for organismal homeostasis. Therefore, understanding how cells generate, shape and maintain lumina is crucial for understanding normal organogenesis as well as the basis of pathological conditions. Lumen formation involves polarized membrane trafficking, cytoskeletal dynamics, and the influence of intracellular as well as extracellular mechanical forces, such as cortical tension, luminal pressure or blood flow. Various tissue culture and in vivo model systems, ranging from MDCK cell spheroids to tubular organs in worms, flies, fish, and mice, have provided many insights into the molecular and cellular mechanisms underlying lumenogenesis and revealed key factors that regulate the size and shape of cellular tubes. Moreover, the development of new experimental and imaging approaches enabled quantitative analyses of intracellular dynamics and allowed to assess the roles of cellular and tissue mechanics during tubulogenesis. However, how intracellular processes are coordinated and regulated across scales of biological organization to generate properly sized and shaped tubes is only beginning to be understood. Here, we review recent insights into the molecular, cellular and physical mechanisms underlying lumen formation during organogenesis. We discuss how these mechanisms control lumen formation in various model systems, with a special focus on the morphogenesis of tubular organs in Drosophila.


Assuntos
Drosophila , Organogênese , Animais , Citoesqueleto , Camundongos , Modelos Biológicos , Morfogênese
18.
Dev Cell ; 56(8): 1083-1099.e5, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33831351

RESUMO

Paracellular permeability is regulated to allow solute transport or cell migration across epithelial or endothelial barriers. However, how cell-cell junction dynamics controls paracellular permeability is poorly understood. Here, we describe patency, a developmentally regulated process in Drosophila oogenesis, during which cell vertices in the follicular epithelium open transiently to allow paracellular transport of yolk proteins for uptake by the oocyte. We show that the sequential removal of E-cadherin, N-cadherin, NCAM/Fasciclin 2, and Sidekick from vertices precedes their basal-to-apical opening, while the subsequent assembly of tricellular occluding junctions marks the termination of patency and seals the paracellular barrier. E-cadherin-based adhesion is required to limit paracellular channel size, whereas stabilized adherens junctions, prolonged NCAM/Fasciclin 2 expression, blocked endocytosis, or increased actomyosin contractility prevent patency. Our findings reveal a key role of cell vertices as gateways controlling paracellular transport and demonstrate that dynamic regulation of adhesion and actomyosin contractility at vertices governs epithelial barrier properties.


Assuntos
Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Oogênese , Folículo Ovariano/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Animais , Transporte Biológico , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Permeabilidade da Membrana Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Endocitose , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Junções Íntimas/metabolismo , Vitelogênese
19.
PLoS Biol ; 5(6): e145, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17503969

RESUMO

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. We isolated mutations in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, we noted an expanded expression pattern of the Krüppel (Kr) and knirps (kni) genes. We found that Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. We show that both Drosophila and human Brakeless and Atrophin interact in vitro, and propose that they act together as a co-repressor complex in many developmental contexts. We discuss the possibility that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA).


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Crescimento Neural/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Dados de Sequência Molecular , Fatores de Crescimento Neural/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos
20.
Curr Biol ; 30(21): 4254-4262.e5, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32857972

RESUMO

Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained, and remodeled is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8, 9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10, 11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here, we show that M6 is essential for the assembly of tricellular, but not bicellular, occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Proteínas da Mielina/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Proteolipídeos/metabolismo , Receptores Depuradores/metabolismo , Junções Íntimas/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Recuperação de Fluorescência Após Fotodegradação , Microscopia Intravital , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas da Mielina/genética , Proteína Proteolipídica de Mielina/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteolipídeos/genética , Receptores Depuradores/genética
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