Thierry Galli

French Institute of Health and Medical Research | Inserm · Institute of Psychiatry and Neurosciences of Paris

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular c...

Intracellular membrane protein trafficking is crucial for both normal cellular physiology and cell-cell communication. The conventional secretory route follows transport from the Endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus. Alternative modes of secretion which can bypass the need for passage through the Golgi apparatus...

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 st...

The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson’s disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the...

Twenty years of research have led to the model, acknowledged by the 2013 Nobel Prize in Physiology and Medicine, that soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the fusion of intracellular membranes. SNAREs are also able to recruit other molecules playing essential roles in the spatial and temporal regul...

Autophagy is being involved in an increasing number of cellular pathways. It now appears that autophagy stimulation and inhibition have complex effects in neurons. Here, we present a simple yet powerful protocol to induce autophagy in primary neurons in culture by partial nutrient deprivation, in neurons with or without transfection of plasmids enc...

Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant...

The compartmentalisation achieved by confining cytoplasm into membrane-enclosed organelles in eukaryotic cells is essential for maintaining vital functions including ATP production, synthetic and degradative pathways. While intracellular organelles are highly specialised in these functions, the restricting membranes also impede exchange of molecule...

Together with the proteasome, macroautophagy is a main pathway for the degradation of intracellular elements. Endoplasmic reticulum (ER)-autophagy i.e. reticulophagy/ER-phagy leads to the encapsulation of pieces of the ER in forming autophagosomes. This is generally followed by fusion with lysosomes and degradation of these ER components by lysosom...

Inter-organelle communication is essential for the exchange of cellular content in eukaryotes, particularly at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM). Accomplishing this critical task requires close positioning of the involved membranes via tether proteins and associated complexes. One such comple...

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this pro...

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b–Stx1 SNARE complexes at endoplasmic reticulum (ER)–PM contacts. Here, we show that Sec22b interacts with members of the extended synaptota...

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b/Stx1 SNARE complexes at endoplasmic reticulum (ER)/PM contacts. Here we show that Sec22b interacts with the endoplasmic reticulum lipid tr...

The neuronal serine protease tissue-type Plasminogen Activator (tPA) is an important player of the neuronal survival and of the synaptic plasticity. Thus, a better understanding the mechanisms regulating the neuronal trafficking of tPA is required to further understand how tPA can influence brain functions. Using confocal imaging including living c...

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specifi...

This dataset includes uncropped blots for all experimental replicates that are represented in Figure 1. Treatments and immunoblot methods were performed as outlined in Figure 1. Blots were probed with indicated anti-VAMP7 antibodies and anti-α-tubulin antibodies was used as a loading control. (A) Dataset used for Figure 1, with cropped regions in r...

Raw images of additional experimental replicates for Figure 2, immunofluorescence experiments This dataset includes additional images from experimental replicates of the images presented in Figure 2. Immunofluorescence staining was performed as described for Figure 2. Images were taken at 40× objective. Bar, 15µm.

Uncropped data from Figure 3 (A) and replicate (C) for VAMP7 immunoprecipitation from Cos-7 cell lysate overexpressing GFP-tagged mouse, rat or human VAMP7 constructs. Uncropped immunoblotting data from Figure 3 (B) and replicate (D) for VAMP7 immunoprecipitation from WT and VAMP7 KO mouse cortex extracts. Antibodies used for immunoprecipitation an...

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright, a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono- and two-photon m...

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specifi...

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon mi...

Effect of siRNA-mediated AP-3μ1 depletion on sealing zone dynamics illustrated by time-lapse video microscopy shown in Figure 4B. The dynamics of sealing zones visualized with mRFP-ezrin actin-binding domain in osteoclasts grown on apatite-collagen-coated (ACC) surfaces.

Dynamics of ARAP1 at sealing zones visualized with the GFP-ARAP1 and with the mRFP-ezrin actin-binding domain in osteoclasts grown on apatite-collagen-coated (ACC) surfaces shown in Figure S1 (200 ms per frame, 1 frame per minute for 30 min).

Video corresponding to Figure 4B. The dynamics of sealing zones visualized with mRFP-ezrin actin-binding domain in osteoclasts grown on apatite-collagen-coated (ACC) surfaces.

Dynamics of ARAP1 at podosomes visualized with the GFP-ARAP1 and with the mRFP-ezrin actin-binding domain in osteoclasts grown on glass coverslips shown in Figure S1. (100 ms per frame, 1 frame per 2 s).

Effect of siRNA-mediated ARAP1 depletion on sealing zone dynamics illustrated by time-lapse video microscopy shown in Figure 4B. The dynamics of sealing zones visualized with mRFP-ezrin actin-binding domain in osteoclasts grown on apatite-collagen-coated (ACC) surfaces.

Bone-resorbing osteoclasts play a central role in bone remodeling and its pathology. To digest bone, osteoclasts re-organize both F-actin, to assemble podosomes/sealing zones, and membrane traffic, to form bone-facing ruffled borders enriched in lysosomal membrane proteins. It remains elusive how these processes are coordinated. Here, we show that...

A cell is able to sense the biomechanical properties of the environment such as the rigidity of the extracellular matrix and adapt its tension via regulation of plasma membrane and underlying actomyosin meshwork properties. The cell’s ability to adapt to the changing biomechanical environment is important for cellular homeostasis and also cell dyna...

Video S1. pHluorin Imaging Reveals Exocytic Fusion Events on the Ventral Cell Membrane, Related to Figure 3 A representative COS7 cell expressing VAMP7-pHluorin and imaged by wide field microscopy 24 hr after plating on laminin-coated 28-kPa PDMS gel. Scale bar: 10 μm, time in seconds.

Video S3. Hyper-Osmotic Pressure Could Instantaneously and Reversibly Slow Down Exocytosis Frequency of VAMP7, Related to Figure 4 A representative COS7 cell expressing pHluorin-tagged ΔLongin-VAMP7 and imaged by wide field microscopy 24 hr after plating on laminin-coated 28-kPa PDMS gel. Images were processed to increase visibility of fusion even...

Fusion events were indicated by a white circle. Scale bar: 5 μm, time in seconds.

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we show that cells deficient in the secretory lysosome v-SNARE VAMP7 are impaired in adaptation to substrate rigidity....

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are...

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are...

Autophagy breaks down nonessential cellular components to replenish macromolecular building blocks during starvation. Nevertheless, the downstream events regulating vesicle trafficking during this essential cellular process are not yet fully defined. Xuet al.combined approaches of crystallography, biochemistry, and cell biology to show that the gua...

RFP-VAMP7 containing vesicles dynamics in control (left) and SPG4-KO (right) 5-DIV cortical neurons. Cortical neurons were transfected for 3h with RFP-VAMP7 at 4-DIV and imaged live 16h after transfection. Time is indicated in min:sec. Acceleration 5×. Scale bar, 20 um.

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders as well as diseases including obesity, diabetes and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenin...

Restoring antigen presentation for efficient and durable activation of tumor-specific CD8+ T-cell responses is pivotal to immunotherapy, yet the mechanisms that cause subversion of dendritic cell (DC) functions are not entirely understood, limiting the development of targeted approaches. In this study, we show that bona fide DCs resident in lung tu...

Intracellular membrane trafficking in eukaryotes is a multiple step process consisting in the budding of vesicles from a donor compartment, their translocation into the cytoplasm along cytoskeletal elements, their tethering, and subsequent fusion with the membrane of the target compartment. Membrane fusion is based on SNARE proteins, classified int...

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we found that cells deficient in the secretory lysosome v-SNARE VAMP7 were impaired in adapting to substrate rigidity....

Alteration of axonal transport has emerged as a common precipitating factor in several neurodegenerative disorders including Human Spastic Paraplegia (HSP). Mutations of the SPAST (SPG4) gene coding for the spastin protein account for 40% of all autosomal dominant uncomplicated HSP. By cleaving microtubules, spastin regulates several cellular proce...

Twenty years of research have led to the model, acknowledged by the 2013 Nobel Prize in Physiology and Medicine, that soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the fusion of intracellular membranes. SNAREs are also able to recruit other molecules playing essential roles in the spatial and temporal regul...

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target SNAREs are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the p...

Inside eukaryotic cells, membrane contact sites (MCSs), regions where two membrane-bound organelles are apposed at less than 30 nm, generate regions of important lipid and calcium exchange. This review principally focuses on the structure and the function of MCSs between the endoplasmic reticulum (ER) and the plasma membrane (PM). Here we describe...

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-...

Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesic...

Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesic...

Movie S4. VAMP-7/IL-12 Vesicles Jointly Reach the DC-IS Membrane, Related to Figure 5E Time-lapse confocal microscopy of DCs co-transfected with p35-GFP and VAMP7-RFP, forming a conjugate with a T cell (blu). Frames were taken every 30 s for 30 min.

Time-lapse confocal microscopy of VAMP7-RFP expressing DC during formation of conjugates with naive T cells stained with membrane dye Bodiby630 (blu). The movie shows that cluster of VAMP7 polarize toward the interaction plane upon T cell contact. Interestingly, VAMP7 transiently moved away from the first T cell to travel towards a second T cell co...

Movie S2. Swarms of IL-12 p35 Vesicles Gather underneath the DC-IS Membrane, Related to Figure 5D Epifluorescence imaging of DC transfected with p35-GFP in synapse with a CD4+ T cell. The position of the T cell is indicated by white dotted lines. Imaging was started short after contact formation. The time-lapse series show bright, fast-moving dots...

Movie S3. Docking of IL-12 p35 Vesicles at the DC-IS Membrane, Related to Figure 5D High magnification of Movie S2 in the synaptic region shows p35+ tubules docking at the PM.

The cation channel TRPM8 plays a central role in the somatosensory system, as a key sensor of innocuously cold temperatures and cooling agents. Although increased functional expression of TRPM8 has been implicated in various forms of pathological cold hypersensitivity, little is known about the cellular and molecular mechanisms that determine TRPM8...

Brain formation requires the establishment of complex neural circuits between a diverse array of neuronal subtypes in an intricate and ever changing micro-environment and yet with a large degree of specificity and reproducibility. In the last three decades, mounting evidence has established that neuronal development relies on the coordinated regula...

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite thatexploits different host vesicular pathways to invade the target cells. Vesicular and target solubleN‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) are key proteins of theintracellular membrane fusion machinery. During the earl...

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the core membrane fusion machinery of intracellular transport and intercellular communication. A little more than ten years ago, it was proposed that the long N-terminal domain of a subset of SNAREs, henceforth called the longin domain, could be a cruc...

Botulinum neurotoxins are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs. However, their serotype A (BoNT/A) which cleaves synaptosomal associated protein of 25 kDa (SNAP-25) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivi...