Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2.

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function

Review article

In eukaryotic cells, the trans-Golgi network (TGN) serves as a platform for secretory cargo sorting and trafficking. In recent years, it has become evident that a complex network of lipid-lipid and lipid-protein interactions contributes to these key functions. This review addresses the role of lipids at the TGN with a particular emphasis on sphingolipids and diacylglycerol. We further highlight how these lipids couple secretory cargo sorting and trafficking for spatiotemporal coordination of protein transport to the plasma membrane

TFEB controls integrin-mediated endothelial cell adhesion by the regulation of cholesterol metabolism

The dynamic integrin-mediated adhesion of endothelial cells (ECs) to the surrounding ECM is fundamental for angiogenesis both in physiological and pathological conditions, such as embryonic development and cancer progression. The dynamics of EC-to-ECM adhesions relies on the regulation of the conformational activation and trafficking of integrins. Here, we reveal that oncogenic transcription factor EB (TFEB), a known regulator of lysosomal biogenesis and metabolism, also controls a transcriptional program that influences the turnover of ECM adhesions in ECs by regulating cholesterol metabolism. We show that TFEB favors ECM adhesion turnover by promoting the transcription of genes that drive the synthesis of cholesterol, which promotes the aggregation of caveolin-1, and the caveolin-dependent endocytosis of integrin β1. These findings suggest that TFEB might represent a novel target for the pharmacological control of pathological angiogenesis and bring new insights in the mechanism sustaining TFEB control of endocytosis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10456-022-09840-x

Exploración del papel de rutas principales de señalización en la morfogénesis epitelial

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-10-2019Una de las cuestiones centrales en biología del desarrollo es cómo los órganos adquieren su forma y tamaño final. Hay dos tipos de comunicación principales que controlan el desarrollo de los órganos: las interacciones biofísicas y la comunicación célula-célula a través de morfógenos. Las señales mecánicas guían diferentes procesos, incluyendo motilidad, crecimiento y diferenciación celular, y los gradientes de morfógenos tienen un papel crítico en el modelado y el crecimiento del órgano. Hasta qué punto son los dos tipos de comunicación necesarios y cuáles son los componentes moleculares específicos que aseguran una adecuada organogénesis son factores que cambian dependiendo del órgano y el estadío de desarrollo. En esta tesis investigamos el papel potencial de varias rutas y de la señalización mecánica de las células mesenquimales durante la tubulogénesis de diferentes órganos epiteliales, como la glándula mamaria y el intestino de pez cebra. Usamos el disco imaginal de ala de Drosophila para entender el mecanismo por el cual Sfrp3 podría estar modulando la ruta de señalización Wnt durante el desarrollo de la glándula mamaria, lo que nos llevó a la conclusión de que SFRP3 no está actuando como regulador negativo de la actividad Wnt sino como difusor de los ligandos Wnt. Empleando técnicas de secuenciación de nueva generación, identificamos que los peces cebra que portan una mutación en el transductor de la ruta Hedgehog smoothened (smo), los cuales presentan defectos previamente descritos en la formación de un único lumen en el intestino, muestran diferentes perfiles transcripcionales en sus células epiteliales intestinales comparados con los peces control . Además, también mostramos que la inhibición de la ruta TGF-β genera un defecto en la resolución de lúmenes en el intestino de embriones de pez cebra en desarrollo. Usando diferentes líneas de pez cebra, intentamos entender las causas de este fenotipo y su posible relación con el fenotipo observado en los mutantes smo. A su vez mostramos cómo la migración de las células mesenquimales alrededor del tubo epitelial está afectada por la inhibición de TGF-β, siendo la falta de restricciones físicas una posible explicación para el fenotipo observado en el caso de la falta de señalización TGF-β. Finalmente, también demostramos que la inhibición de TGF-β altera la morfogénesis epitelial de esferoides MDCK in vitro, y que TGF-β podría estar regulando la orientación del huso mitótico afectando a la maquinaria que controla este proceso.Este trabajo ha sido financiado por subvenciones del MINECO (BFU2014--‐57831;BFU2015--‐71244) y la Fundación Ramón Arece

Virus:host interactions during chikungunya virus infection:Analyzing host cell factors and antiviral strategies

In the past decades we have witnessed a drastic re-emergence of mosquito-borne viruses. While millions of people are at risk to become infected there are no antiviral therapies or vaccines available to treat or prevent infection by these viruses. One of the rapidly spreading mosquito-borne viruses is chikungunya virus. Infection with chikungunya virus can lead to a debilitating illness including flu-like symptoms, headache, and long-lasting rheumatic disease symptoms. To develop antiviral therapies, it is a prerequisite to understand the virus:host interactions that lead to disease. In this thesis we investigated the virus:host interactions required for chikungunya virus replication. Emphasis was on identifying new cellular factors that are important for chikungunya virus infection and to explore whether these might serve as targets for antiviral therapeutics. We analyzed the role of the cellular receptor Mxra8 and microtubules in the early events of chikungunya virus infection. Moreover, we studied the antiviral activity of serotonergic drugs, heat shock protein inhibitors and the neutralizing antibody CHK-152. Overall, these research activities provide future perspectives on the cellular factors required for chikungunya virus infection that could be targeted for anti-chikungunya virus drug development

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana

Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

Background Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t \u3e ~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%. Conclusions The mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport

Chikungunya virus requires an intact microtubule network for efficient viral genome delivery

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating

The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in Normal Rat Kidney cells

Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family