Quality assessment in light microscopy for routine use through simple tools and robust metrics.

Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.

Δ133p53ß isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells.

The p53 isoform, Δ133p53ß, is critical in promoting cancer. Here we report that Δ133p53ß activity is regulated through an aggregation-dependent mechanism. Δ133p53ß aggregates were observed in cancer cells and tumour biopsies. The Δ133p53ß aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53ß aggregates and loss of Δ133p53ß dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53ß from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion.

Imaging translation dynamics in live embryos reveals spatial heterogeneities.

Much is known about the factors involved in the translation of messenger RNA (mRNA) into protein; however, this multistep process has not been imaged in living multicellular organisms. Here, we deploy the SunTag method to visualize and quantify the timing, location, and kinetics of the translation of single mRNAs in living Drosophila embryos. By focusing on the translation of the conserved major epithelial-mesenchymal transition-inducing transcription factor Twist, we identify spatial heterogeneity in mRNA translation efficiency and reveal the existence of translation factories, where clustered mRNAs are cotranslated preferentially at basal perinuclear regions. Observing the location and dynamics of mRNA translation in a living multicellular organism opens avenues for understanding gene regulation during development.

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ.

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.

Tensin 3 is a new partner of Dock5 that controls osteoclast podosome organization and activity.

Bone resorption by osteoclasts is mediated by a typical adhesion structure called the sealing zone or actin ring, whose architecture is based on a belt of podosomes. The molecular mechanisms driving podosome organization into superstructures remain poorly understood to date, in particular at the osteoclast podosome belt. We performed proteomic analyses in osteoclasts and found that the adaptor protein tensin 3 is a partner of Dock5, a Rac exchange factor necessary for podosome belt formation and bone resorption. Expression of tensin 3 and Dock5 concomitantly increase during osteoclast differentiation. These proteins associate with the osteoclast podosome belt but not with individual podosomes, in contrast to vinculin. Super-resolution microscopy revealed that, even if they colocalize in the x-y plane of the podosome belt, Dock5 and tensin 3 differentially localize relative to vinculin in the z-axis. Tensin 3 increases Dock5 exchange activity towards Rac, and suppression of tensin 3 in osteoclasts destabilizes podosome organization, leading to delocalization of Dock5 and a severe reduction in osteoclast activity. Our results suggest that Dock5 and tensin 3 cooperate for osteoclast activity, to ensure the correct organization of podosomes.

The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth.

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5(-/-) mice have increased trabecular bone mass. We used Dock5(-/-) mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5(-/-) mice. We demonstrate that Dock5(-/-) mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5(-/-) growing mice as compared to Dock5(+/+) animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5(-/-) osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.

Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion.

Cadherins are homophilic cell-cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell-cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell-cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell-cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell-cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2-positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell-cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion.