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Ultrastructural characteristics of finger-like membrane protrusions in cell competition.

ABSTRACT: A small number of oncogenic mutated cells sporadically arise within the epithelial monolayer. Newly emerging Ras- or Src-transformed epithelial cells are often apically eliminated during competitive interactions between normal and transformed cells. Our recent electron microscopy (EM) analyses revealed that characteristic finger-like membrane protrusions are formed at the interface between normal and RasV12-transformed cells via the cdc42-formin-binding protein 17 (FBP17) pathway, potentially playing a positive role in intercellular recognition during apical extrusion. However, the spatial distribution and ultrastructural characteristics of finger-like protrusions remain unknown. In this study, we performed both X-Y and X-Z EM analyses of finger-like protrusions during the apical extrusion of RasV12-transformed cells. Quantification of the distribution and widths of the protrusions showed comparable results between the X-Y and X-Z sections. Finger-like protrusions were observed throughout the cell boundary between normal and RasV12 cells, except for apicalmost tight junctions. In addition, a non-cell-autonomous reduction in protrusion widths was observed between RasV12 cells and surrounding normal cells under the mix culture condition. In the finger-like protrusions, intercellular adhesions via thin electron-dense plaques were observed, implying that immature and transient forms of desmosomes, adherens junctions or unknown weak adhesions were distributed. Interestingly, unlike RasV12-transformed cells, Src-transformed cells form fewer evident protrusions, and FBP17 in Src cells is dispensable for apical extrusion. Collectively, these results suggest that the dynamic reorganization of intercellular adhesions via finger-like protrusions may positively control cell competition between normal and RasV12-transformed cells. Furthermore, our data indicate a cell context-dependent diversity in the modes of apical extrusion.

SUBMITTER: Kamasaki T

PROVIDER: S-EPMC9340795 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Ultrastructural characteristics of finger-like membrane protrusions in cell competition.

Kamasaki Tomoko T Uehara Ryota R Fujita Yasuyuki Y

Microscopy (Oxford, England) 20220801 4

A small number of oncogenic mutated cells sporadically arise within the epithelial monolayer. Newly emerging Ras- or Src-transformed epithelial cells are often apically eliminated during competitive interactions between normal and transformed cells. Our recent electron microscopy (EM) analyses revealed that characteristic finger-like membrane protrusions are formed at the interface between normal and RasV12-transformed cells via the cdc42-formin-binding protein 17 (FBP17) pathway, potentially pl ...[more]

PMID: 35394538

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Project description:Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the E. histolytica surface metalloprotease EhMSP-1 produces hyperadherent less-motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1-silenced phenotype, here we compared the phosphoproteomes of EhMSP-1-silenced and vector control trophozoites by using quantitative tandem mass spectrometry-based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1-silenced and control cells, including EhCoactosin, a member of the ADF/cofilin family of actin-binding proteins, which was more frequently phosphorylated at serine 147. Regulated overexpression of wild-type, phosphomimetic, and nonphosphorylatable EhCoactosin variants was used to test if phosphorylation functions in control of E. histolytica actin dynamics. Each of the overexpressed proteins colocalized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing an EhCoactosin phosphomimetic mutant formed more and poorly coordinated cell membrane protrusions compared to those in control or cells expressing a nonphosphorylatable mutant, while trophozoites overexpressing nonphosphorylatable EhCoactosin were significantly more motile within a model of mammalian extracellular matrix. Therefore, although EhCoactosin's actin-binding ability appeared unaffected by phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1-silenced trophozoites and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness.IMPORTANCE Invasive amoebiasis, caused by the intestinal parasite Entamoeba histolytica, causes life-threatening diarrhea and liver abscesses, but, for unknown reasons, only approximately 10% of E. histolytica infections become symptomatic. A key requirement of invasion is the ability of the parasite to migrate through tissue layers. Here, we systematically looked for differences in protein phosphorylation between control parasites and a previously identified hyperadherent E. histolytica cell line that has reduced motility. We identified EhCoactosin, an actin-binding protein not previously known to be phosphoregulated, as one of the differentially phosphorylated proteins in E. histolytica and demonstrated that EhCoactosin phosphorylation functions in control of cell membrane dynamics and amoebic motility. This and the additional differentially phosphorylated proteins reported lay the groundwork for identifying kinases and phosphatases that regulate tissue invasiveness.

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Ultrastructural characteristics of finger-like membrane protrusions in cell competition.

Publications

Ultrastructural characteristics of finger-like membrane protrusions in cell competition.

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