Biogenesis of specialized lysosomes in differentiated keratinocytes relies on close apposition with the Golgi apparatus.

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling of lysosomes and the Golgi apparatus. However, it was not known whether changes in Golgi and lysosome morphology and their redistribution were linked. Here, we show that disassembled Golgi is distributed in close physical apposition to lysosomes in differentiated keratinocytes. This atypical localization requires the Golgi tethering protein GRASP65, which is associated with both the Golgi and lysosome membranes. Depletion of GRASP65 results in the loss of Golgi-lysosome apposition and the malformation of lysosomes, defined by their aberrant morphology, size, and function. Surprisingly, a trans-Golgi enzyme and secretory Golgi cargoes are extensively localized to the lysosome lumen and secreted to the cell surface, contributing to total protein secretion of differentiated keratinocytes but not in proliferative precursors, indicating that lysosomes acquire specialization during differentiation. We further demonstrate that the secretory function of the Golgi apparatus is critical to maintain keratinocyte lysosomes. Our study uncovers a novel form of Golgi-lysosome cross-talk and its role in maintaining specialized secretory lysosomes in differentiated keratinocytes.

BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

Spatial organization of lysosomal exocytosis relies on membrane tension gradients.

Lysosomal exocytosis is involved in many key cellular processes but its spatiotemporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-ß-cyclodextrin were found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allow to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM), we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs.

Transcription factor EB regulates phosphatidylinositol-3-phosphate levels that control lysosome positioning in the bladder cancer model.

Lysosomes orchestrate degradation and recycling of exogenous and endogenous material thus controlling cellular homeostasis. Little is known how this organelle changes during cancer. Here we investigate the intracellular landscape of lysosomes in a cellular model of bladder cancer. Employing standardized cell culture on micropatterns we identify a phenotype of peripheral lysosome positioning prevailing in bladder cancer cell lines but not normal urothelium. We show that lysosome positioning is controlled by phosphatidylinositol-3-phosphate (PtdIns3P) levels on endomembranes which recruit FYVE-domain containing proteins for lysosomal dispersion. We identify transcription factor EB (TFEB) as an upstream regulator of PtdIns3P production by VPS34 that is activated in aggressive bladder cancer cells with peripheral lysosomes. This conceptually clarifies the dual role of TFEB as regulator of endosomal maturation and autophagy, two distinct processes controlled by PtdIns3P. Altogether, our findings uncover peripheral lysosome positioning, resulting from PtdIns3P production downstream of TFEB activation, as a potential biomarker for bladder cancer.

RAB6 and dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination.

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that aRG delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3 and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knockout of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.

Contributions of Andrée Tixier-Vidal (1923-2021) to modern cell biology.

This article illustrates the main stages of the scientific career of Dr Andrée Tixier-Vidal, a pioneer in cell biology research in France. She made important discoveries in the field of hormone secretion and neuronal morphogenesis. She played a key role in developing pituitary and neuronal cultures and using electron microscopy to study cellular structures. Her scientific influence continues to irradiate through her students and collaborators.

Generating Rab6 Conditional Knockout Mice.

RAB6 GTPase is the most abundant Golgi-associated RAB protein and regulates several transport steps at the level of this organelle. Homozygous Rab6a knockout (k/o) is embryonic lethal in mouse. To study RAB6 function in cell lineages and tissues, we thus generated various conditional Rab6a knockout (k/o) mice using the Cre/lox system.

Multiscale rheology of glioma cells.

Cells tend to soften during cancer progression, suggesting that mechanical phenotyping could be used as a diagnostic or prognostic method. Here we investigate the cell mechanics of gliomas, brain tumors that originate from glial cells or glial progenitors. Using two microrheology techniques, a single-cell parallel plates rheometer to probe whole-cell mechanics and optical tweezers to probe intracellular rheology, we show that cell mechanics discriminates human glioma cells of different grades. When probed globally, grade IV glioblastoma cells are softer than grade III astrocytoma cells, while they are surprisingly stiffer at the intracellular level. We explain this difference between global and local intracellular behaviours by changes in the composition and spatial organization of the cytoskeleton, and by changes in nuclear mechanics. Our study highlights the need to combine rheology techniques for potential diagnostic or prognostic methods based on cancer cell mechanophenotyping.

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types.

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.

Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway.

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.

RAB6 GTPase regulates mammary secretory function by controlling the activation of STAT5.

The Golgi-associated RAB GTPases, RAB6A and RAB6A', regulate anterograde and retrograde transport pathways from and to the Golgi. In vitro, RAB6A/A' control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described in vivo Here, we generated BlgCre; Rab6aF/F mice presenting a specific deletion of Rab6a in the mammary luminal secretory lineage during gestation and lactation. Rab6a loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon Rab6a deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A' in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A' depend on cell-type specialization and tissue context.

Ruthenium(II) Complex Containing a Redox-Active Semiquinonate Ligand as a Potential Chemotherapeutic Agent: From Synthesis to In Vivo Studies.

Chemotherapy remains one of the dominant treatments to cure cancer. However, due to the many inherent drawbacks, there is a search for new chemotherapeutic drugs. Many classes of compounds have been investigated over the years to discover new targets and synergistic mechanisms of action including multicellular targets. In this work, we designed a new chemotherapeutic drug candidate against cancer, namely, [Ru(DIP)2(sq)](PF6) (Ru-sq) (DIP = 4,7-diphenyl-1,10-phenanthroline; sq = semiquinonate ligand). The aim was to combine the great potential expressed by Ru(II) polypyridyl complexes and the singular redox and biological properties associated with the catecholate moiety. Experimental evidence (e.g., X-ray crystallography, electron paramagnetic resonance, electrochemistry) demonstrates that the semiquinonate is the preferred oxidation state of the dioxo ligand in this complex. The biological activity of Ru-sq was then scrutinized in vitro and in vivo, and the results highlight the promising potential of this complex as a chemotherapeutic agent against cancer.

Synthesis, Characterization, Cytotoxic Activity, and Metabolic Studies of Ruthenium(II) Polypyridyl Complexes Containing Flavonoid Ligands.

Four novel monocationic Ru(II) polypyridyl complexes were synthesized with the general formula [Ru(DIP)2flv]X, where DIP is 4,7-diphenyl-1,10-phenanthroline, flv stands for the flavonoid ligand (5-hydroxyflavone in [Ru(DIP)2(5-OHF)](PF6), genistein in [Ru(DIP)2(gen)](PF6), chrysin in [Ru(DIP)2(chr)](OTf), and morin in [Ru(DIP)2(mor)](OTf)), and X is the counterion, PF6-, and OTf ̅ (triflate, CF3SO3̅), respectively. Following the chemical characterization of the complexes by 1H and 13C NMR, mass spectrometry, and elemental analysis, their cytotoxicity was tested against several cancer cell lines. The most promising complex, [Ru(DIP)2(gen)](PF6), was further investigated for its biological activity. Metabolic studies revealed that this complex severely impaired mitochondrial respiration and glycolysis processes, contrary to its precursor, Ru(DIP)2Cl2, which showed a prominent effect only on the mitochondrial respiration. In addition, its preferential accumulation in MDA-MB-435S cells (a human melanoma cell line previously described as mammary gland/breast; derived from metastatic site: pleural effusion), which are used for the study of metastasis, explained the better activity in this cell line compared to MCF-7 (human, ductal carcinoma).

Rationally Designed Long-Wavelength Absorbing Ru(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy.

The utilization of photodynamic therapy (PDT) for the treatment of various types of cancer has gained increasing attention over the last decades. Despite the clinical success of approved photosensitizers (PSs), their application is sometimes limited due to poor water solubility, aggregation, photodegradation, and slow clearance from the body. To overcome these drawbacks, research efforts are devoted toward the development of metal complexes and especially Ru(II) polypyridine complexes based on their attractive photophysical and biological properties. Despite the recent research developments, the vast majority of complexes utilize blue or UV-A light to obtain a PDT effect, limiting the penetration depth inside tissues and, therefore, the possibility to treat deep-seated or large tumors. To circumvent these drawbacks, we present the first example of a DFT guided search for efficient PDT PSs with a substantial spectral red shift toward the biological spectral window. Thanks to this design, we have unveiled a Ru(II) polypyridine complex that causes phototoxicity in the very low micromolar to nanomolar range at clinically relevant 595 nm, in monolayer cells as well as in 3D multicellular tumor spheroids.

Increasing the Cytotoxicity of Ru(II) Polypyridyl Complexes by Tuning the Electronic Structure of Dioxo Ligands.

Due to the great potential expressed by an anticancer drug candidate previously reported by our group, namely, Ru-sq ([Ru(DIP)2(sq)](PF6) (DIP, 4,7-diphenyl-1,10-phenanthroline; sq, semiquinonate ligand), we describe in this work a structure-activity relationship (SAR) study that involves a broader range of derivatives resulting from the coordination of different catecholate-type dioxo ligands to the same Ru(DIP)2 core. In more detail, we chose catechols carrying either an electron-donating group (EDG) or an electron-withdrawing group (EWG) and investigated the physicochemical and biological properties of their complexes. Several pieces of experimental evidences demonstrated that the coordination of catechols bearing EDGs led to deep-red positively charged complexes 1-4 in which the preferred oxidation state of the dioxo ligand is the uninegatively charged semiquinonate. Complexes 5 and 6, on the other hand, are blue/violet neutral complexes, which carry an EWG-substituted dinegatively charged catecholate ligand. The biological investigation of complexes 1-6 led to the conclusion that the difference in their physicochemical properties has a strong impact on their biological activity. Thus, complexes 1-4 expressed much higher cytotoxicities than complexes 5 and 6. Complex 1 constitutes the most promising compound in the series and was selected for a more in depth biological investigation. Apart from its remarkably high cytotoxicity (IC50 = 0.07-0.7 µM in different cancerous cell lines), complex 1 was taken up by HeLa cells very efficiently by a passive transportation mechanism. Moreover, its moderate accumulation in several cellular compartments (i.e., nucleus, lysosomes, mitochondria, and cytoplasm) is extremely advantageous in the search for a potential drug with multiple modes of action. Further DNA metalation and metabolic studies pointed to the direct interaction of complex 1 with DNA and to the severe impairment of the mitochondrial function. Multiple targets, together with its outstanding cytotoxicity, make complex 1 a valuable candidate in the field of chemotherapy research. It is noteworthy that a preliminary biodistribution study on healthy mice demonstrated the suitability of complex 1 for further in vivo studies.

A Maltol-Containing Ruthenium Polypyridyl Complex as a Potential Anticancer Agent.

Cancer is one of the main causes of death worldwide. Chemotherapy, despite its severe side effects, is to date one of the leading strategies against cancer. Metal-based drugs present several potential advantages when compared to organic compounds and they have gained trust from the scientific community after the approval on the market of the drug cisplatin. Recently, we reported the ruthenium complex ([Ru(DIP)2 (sq)](PF6 ) (where DIP is 4,7-diphenyl-1,10-phenantroline and sq is semiquinonate) with a remarkable potential as chemotherapeutic agent against cancer, both in vitro and in vivo. In this work, we analyse a structurally similar compound, namely [Ru(DIP)2 (mal)](PF6 ), carrying the flavour-enhancing agent approved by the FDA, maltol (mal). To possess an FDA approved ligand is crucial for a complex, whose mechanism of action might include ligand exchange. Herein, we describe the synthesis and characterisation of [Ru(DIP)2 (mal)](PF6 ), its stability in solutions and under conditions that resemble the physiological ones, and its in-depth biological investigation. Cytotoxicity tests on different cell lines in 2D model and on HeLa MultiCellular Tumour Spheroids (MCTS) demonstrated that our compound has higher activity than cisplatin, inspiring further tests. [Ru(DIP)2 (mal)](PF6 ) was efficiently internalised by HeLa cells through a passive transport mechanism and severely affected the mitochondrial metabolism.