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1.
Nat Commun ; 15(1): 1277, 2024 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-38341434

RESUMO

Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes. The bridging depends on the PX domains of TKS4/5, which coincidently interact with the cytosolic tail of MT1-MMP and endosomal phosphatidylinositol 3-phosphate. MT1-MMP recruits TKS4/5 into multivesicular endosomes for their subsequent co-secretion in extracellular vesicles, together with the enzymatically active ectodomain. The shed ectodomain converts non-invasive recipient cells into an invasive phenotype. Thus, TKS4/5 promote intercellular transfer of cancer cell invasiveness by facilitating ADAM-mediated shedding of MT1-MMP in acidic endosomes.


Assuntos
Metaloproteinase 14 da Matriz , Neoplasias , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Peptídeo Hidrolases/metabolismo , Neoplasias/genética , Endossomos/metabolismo , Invasividade Neoplásica , Linhagem Celular Tumoral
2.
iScience ; 25(5): 104250, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35521520

RESUMO

Rab proteins are well known regulators of intracellular trafficking; however, more and more studies point to their function also in other cellular processes, including cell migration. In this work, we have performed an siRNA screen to identify Rab proteins that influence cell migration. The screen revealed Rab33b as the strongest candidate that affected cell motility. Rab33b has been previously reported to localize at the Golgi apparatus to regulate Golgi-to-ER retrograde trafficking and Golgi homeostasis. We revealed that Rab33b also mediates post-Golgi transport to the plasma membrane. We further identified Exoc6, a subunit of the exocyst complex, as an interactor of Rab33b. Moreover, our data indicate that Rab33b regulates focal adhesion dynamics by modulating the delivery of cargo such as integrins to focal adhesions. Altogether, our results demonstrate a role for Rab33b in cell migration by regulating the delivery of integrins to focal adhesions through the interaction with Exoc6.

3.
Proc Natl Acad Sci U S A ; 117(46): 28614-28624, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33139578

RESUMO

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III-depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/fisiologia , Modelos Biológicos , Endossomos/ultraestrutura , Células HeLa , Humanos
4.
Nat Cell Biol ; 22(7): 856-867, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601372

RESUMO

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7-LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.


Assuntos
Núcleo Celular/patologia , Cromatina/metabolismo , Aberrações Cromossômicas , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos
5.
J Cell Biol ; 219(7)2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32525992

RESUMO

The members of the Rab family of small GTPases are molecular switches that regulate distinct steps in different membrane traffic pathways. In addition to this canonical function, Rabs can play a role in other processes, such as cell adhesion and motility. Here, we reveal the role of the small GTPase Rab18 as a positive regulator of directional migration in chemotaxis, and the underlying mechanism. We show that knockdown of Rab18 reduces the size of focal adhesions (FAs) and influences their dynamics. Furthermore, we found that Rab18, by directly interacting with the endoplasmic reticulum (ER)-resident protein kinectin-1, controls the anterograde kinesin-1-dependent transport of the ER required for the maturation of nascent FAs and protrusion orientation toward a chemoattractant. Altogether, our data support a model in which Rab18 regulates kinectin-1 transport toward the cell surface to form ER-FA contacts, thus promoting FA growth and cell migration during chemotaxis.


Assuntos
Membrana Celular/metabolismo , Quimiotaxia/genética , Retículo Endoplasmático/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/genética , Proteínas rab de Ligação ao GTP/genética , Transporte Biológico , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismo
6.
Cell Mol Life Sci ; 76(13): 2593-2614, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30830239

RESUMO

Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.


Assuntos
Movimento Celular , Embrião não Mamífero/patologia , Neoplasias/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina , Animais , Embrião não Mamífero/metabolismo , Humanos , Microtúbulos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Transporte Proteico , Transdução de Sinais , Células Tumorais Cultivadas , Peixe-Zebra , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/genética
7.
J Cell Sci ; 131(17)2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30111580

RESUMO

Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various in vitro and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endossomos/enzimologia , Endossomos/genética , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Humanos , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
8.
Biochim Biophys Acta Mol Cell Res ; 1865(10): 1397-1409, 2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30021127

RESUMO

The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.

9.
EMBO Rep ; 18(10): 1727-1739, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28835545

RESUMO

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Cisteína Endopeptidases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Relacionadas à Autofagia/genética , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
10.
Commun Integr Biol ; 8(6): e1023492, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-27066171

RESUMO

Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

11.
Glycobiology ; 25(1): 30-41, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25138304

RESUMO

Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted proteoglycans (PGs) displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.


Assuntos
Sulfatos de Condroitina/metabolismo , Complexo de Golgi/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Animais , Transporte Biológico , Polaridade Celular , Sulfatos de Condroitina/química , Cães , Regulação da Expressão Gênica , Proteoglicanas de Heparan Sulfato/química , Heparitina Sulfato/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana Transportadoras/genética , Fosfoadenosina Fosfossulfato/química , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
12.
J Histochem Cytochem ; 60(12): 926-35, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22941419

RESUMO

A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.


Assuntos
Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Proteoglicanas/biossíntese , Animais , Transporte Biológico , Polaridade Celular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cães , Glicosaminoglicanos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Células Madin Darby de Rim Canino , Proteoglicanas/metabolismo
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