Search

The activation of the Ca 2+ -channel Orai1 via the physiological activator stromal interaction molecule 1 (STIM1) requires structural rearrangements within the entire channel complex involving a series of gating checkpoints. Focusing on the gating mechanism operating along the peripheral transmembrane domain (TM) 3/TM4-interface, we report here that some charged substitutions close to the center of TM3 or TM4 lead to constitutively active Orai1 variants triggering nuclear factor of activated T-cell (NFAT) translocation into the nucleus. Molecular dynamics simulations unveil that this gain-of-function correlates with enhanced hydration at peripheral TM-interfaces, leading to increased local structural flexibility of the channel periphery and global conformational changes permitting pore opening. Our findings indicate that efficient dehydration of the peripheral TM-interfaces driven by the hydrophobic effect is critical for maintaining the closed state of Orai1. We conclude that a charge close to the center of TM3 or TM4 facilitates concomitant hydration and widening of peripheral TM interfaces to trigger constitutive Orai1 pore opening to a level comparable to or exceeding that of native activated Orai1., Competing Interests: Competing interests The authors declare no competing interests. Consent to publish All authors have read and approved its submission to this journal., (© 2024. The Author(s).)

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca 2+ concentration and directly binds to the store-operated Ca 2+ channel Orai1 for its activation when Ca 2+ recedes. At high resting ER-Ca 2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca 2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca 2+ entry via Orai1, decreasing the Ca 2+ oscillation frequency as well as store-operated Ca 2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein., Competing Interests: Competing interests statement:The authors declare no competing interest.

The family of stromal interaction molecules (STIM) includes two widely expressed single-pass endoplasmic reticulum (ER) transmembrane proteins and additional splice variants that act as precise ER-luminal Ca 2+ sensors. STIM proteins mainly function as one of the two essential components of the so-called Ca 2+ release-activated Ca 2+ (CRAC) channel. The second CRAC channel component is constituted by pore-forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation. Their activation commonly requires the emptying of the intracellular ER Ca 2+ store. Using their Ca 2+ sensing capabilities, STIM proteins confer this Ca 2+ content-dependent signal to Orai, thereby linking Ca 2+ store depletion to CRAC channel opening. Here we review the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Ca 2+ release-activated Ca 2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca 2+ -sensor STIM1 in the ER membrane and the Ca 2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca 2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca 2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1., (© 2023. The Author(s).)

Stromal interaction molecule 1 (STIM1) resides in the endoplasmic reticulum (ER) membrane and senses luminal calcium (Ca 2+ ) concentration. STIM1 activation involves a large-scale conformational transition that exposes a STIM1 domain termed "CAD/SOAR", - which is required for activation of the calcium channel Orai. Under resting cell conditions, STIM1 assumes a quiescent state where CAD/SOAR is suspended in an intramolecular clamp formed by the coiled-coil 1 domain (CC1) and CAD/SOAR. Here, we present a structural model of the cytosolic part of the STIM1 resting state using molecular docking simulations that take into account previously reported interaction sites between the CC1α1 and CAD/SOAR domains. We corroborate and refine previously reported interdomain coiled-coil contacts. Based on our model, we provide a detailed analysis of the CC1-CAD/SOAR binding interface using molecular dynamics simulations. We find a very similar binding interface for a proposed domain-swapped configuration of STIM1, where the CAD/SOAR domain of one monomer interacts with the CC1α1 domain of another monomer of STIM1. The rich structural and dynamical information obtained from our simulations reveals novel interaction sites such as M244, I409, or E370, which are crucial for STIM1 quiescent state stability. We tested our predictions by electrophysiological and Förster resonance energy transfer experiments on corresponding single-point mutants. These experiments provide compelling support for the structural model of the STIM1 quiescent state reported here. Based on transitions observed in enhanced-sampling simulations paired with an analysis of the quiescent STIM1 conformational dynamics, our work offers a first atomistic model for CC1α1-CAD/SOAR detachment., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca 2+ sensor protein STIM1, which forms the Ca 2+ release-activated Ca 2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca 2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca 2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu 296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu 296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

In humans, there are three paralogs of the Orai Ca 2+ channel that form the core of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still under active investigation, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating checkpoints among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the "ANSGA" nexus mutations in TM4 of human Orai1, which is reported to mimic the STIM1-activated state of the channel. In investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, that are not conserved among paralogs and that seem to be crucial for the channel activation triggered by the "ANSGA" mutations in Orai1. However, mutations of the same residues still allow gating of Orai1 by STIM1, suggesting that the ANSGA mutant of Orai1 may not be a surrogate for the STIM1-activated state of the Orai1 channel. Our results shed new light on these important gating checkpoints and show that the gating mechanism of Orai channels is affected by multiple factors that are not necessarily conserved among orai homologs, such as the TM4-TM3 coupling., (Copyright © 2022. Published by Elsevier Ltd.)

All human life starts with a calcium (Ca 2+ ) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca 2+ network are store-operated calcium channels in the cells' membrane. The best-characterised store-operated channel is the Ca 2+ release-activated Ca 2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca 2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca 2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca 2+ and its role in human health and disease with focus on CRAC channels.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). This respiratory illness was declared a pandemic by the world health organization (WHO) in March 2020, just a few weeks after being described for the first time. Since then, global research effort has considerably increased humanity's knowledge about both viruses and disease. It has also spawned several vaccines that have proven to be key tools in attenuating the spread of the pandemic and severity of COVID-19. However, with vaccine-related skepticism being on the rise, as well as breakthrough infections in the vaccinated population and the threat of a complete immune escape variant, alternative strategies in the fight against SARS-CoV-2 are urgently required. Calcium signals have long been known to play an essential role in infection with diverse viruses and thus constitute a promising avenue for further research on therapeutic strategies. In this review, we introduce the pivotal role of calcium signaling in viral infection cascades. Based on this, we discuss prospective calcium-related treatment targets and strategies for the cure of COVID-19 that exploit viral dependence on calcium signals.

The interplay of SK3, a Ca 2+ sensitive K + ion channel, with Orai1, a Ca 2+ ion channel, has been reported to increase cytosolic Ca 2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K + currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca 2+ levels that decrease SK3 K + permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3-CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3-Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3-Orai1 interplay by significantly decreasing their co-localization. Forced STIM1-Orai1 activity and associated Ca 2+ influx promote SK3 K + currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1 H, 13 C, 15 N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form., (© 2021. The Author(s).)

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca 2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca 2+ ion channel in the plasma membrane. Ca 2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Legend: Upon intracellular Ca 2+ store depletion of the endoplasmic reticulum (ER), Ca 2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca 2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca 2+ -selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca 2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca 2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right)., (© 2021. The Author(s).)

STIM1-mediated activation of calcium selective Orai channels is fundamental for life. The three Orai channel isoforms, Orai1-3, together with their multiple ways of interplay, ensure their highly versatile role in a variety of cellular functions and tissues in both, health and disease. While all three isoforms are activated in a store-operated manner by STIM1, they differ in diverse biophysical and structural properties. In the present study, we provide profound evidence that non-conserved residues in TM3 control together with the cytosolic loop2 region the maintenance of the closed state and the configuration of an opening-permissive channel conformation of Orai1 and Orai3 in an isoform-specific manner. Indeed, analogous amino acid substitutions of these non-conserved residues led to distinct extents of gain- (GoF) or loss-of-function (LoF). Moreover, we showed that enhanced overall hydrophobicity along TM3 correlates with an increase in GoF mutant currents. Conclusively, while the overall activation mechanisms of Orai channels appear comparable, there are considerable variations in gating checkpoints crucial for pore opening. The elucidation of regions responsible for isoform-specific functional differences provides valuable targets for drug development selective for one of the three Orai homologs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tiffner, Maltan, Fahrner, Sallinger, Weiß, Grabmayr, Höglinger and Derler.)

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α 1 and CC1α 2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.

Store-operated calcium entry (SOCE) through Orai ion channels is an intracellular signaling pathway that is initiated by ligand-induced depletion of calcium from the endoplasmic reticulum (ER) store. The molecular link between SOCE and ER store depletion is thereby provided by a distinct class of single pass ER transmembrane proteins known as stromal interaction molecules (STIM). STIM proteins are equipped with a precise N-terminal calcium sensing domain that enables them to react to changes of the ER luminal calcium concentration. Additionally, a C-terminal coiled-coil domain permits relaying of signals to Orai ion channels via direct physical interaction. In this review, we provide a brief introduction to STIM proteins with a focus on structure and function and give an overview of recent developments in the field of STIM research., Competing Interests: Conflict of interest statement Nothing declared.

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.

Optical super-resolution microscopy is rapidly changing the way imaging studies in the biological and biomedical sciences are conducted. Due to the unique capability of achieving molecular contrast using fluorescent labels and sub-diffraction resolution down to a few tens of nanometers, super-resolution is developing as an attractive imaging modality. While the increased spatial resolution has already enabled structural studies at unprecedented molecular detail, the wide-spread use of super-resolution approaches as a standard characterization technique in biological laboratories has thus far been prevented by mainly two issues: (1) Intricate sample preparation and image acquisition and (2) costly and complex instrumentation. We here introduce a combination of the recently developed super-resolution technique DNA-PAINT (DNA points accumulation for imaging in nanoscale topography) with an easy-to-replicate, custom-built 3D single-molecule microscope (termed liteTIRF) that is an order of magnitude more economic in cost compared to most commercial systems. We assay the performance of our system using synthetic two- and three-dimensional DNA origami structures and show the applicability to single- and multiplexed cellular imaging., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.

Although it is well established that the protein α-synuclein (αS) plays an important role in Parkinson's disease, its physiological function remains largely unknown. It has been reported to bind membranes and to play a role in membrane remodeling processes. The mechanism by which αS remodels membranes is still debated; it may either affect its physical properties or act as a chaperone for other membrane associated proteins. To obtain insight into the role of αS in membrane remodeling we investigated the number of αS proteins associated with single small vesicles in a neuronal cell model. Using single-molecule microscopy and photo-bleaching approaches, we most frequently found 70 αS-GFPs per vesicle. Although this number is high enough to modulate physical membrane properties, it is also strikingly similar to the number of synaptobrevins, a putative interaction partner of αS, per vesicle. We therefore hypothesize a dual, synergistic role for αS in membrane remodeling.

We developed an impedance quartz crystal microbalance (QCM) approach with the ability to simultaneously record mass changes and calibrated energy dissipation with high sensitivity using an impedance analyzer. This impedance QCM measures frequency shifts and resistance changes of sensing quartz crystals very stable, accurately, and calibrated, thus yielding quantitative information on mass changes and dissipation. Resistance changes below 0.3 Ω were measured with corresponding dissipation values of 0.01 µU (micro dissipation units). The broadband impedance capabilities allow measurements between 20 Hz and 120 MHz including higher harmonic modes of up to 11th order for a 10 MHz fundamental resonance frequency quartz crystal. We demonstrate the adsorbed mass, calibrated resistance, and quantitative dissipation measurements on two biological systems including the high affinity based avidin-biotin interaction and nano-assemblies of polyelectrolyte layers. The binding affinity of a protein-antibody interaction was determined. The impedance QCM is a versatile and simple method for accurate and calibrated resistance and dissipation measurements with broadband measurement capabilities for higher harmonics measurements.

The formation of α-synuclein (α-S) amyloid aggregates, called Lewy bodies (LBs), is a hallmark of Parkinson's disease (PD). The function of LBs in the disease process is however still unclear; they have been associated with both neuroprotection and toxicity. To obtain insight into this contradiction, we induced the formation of α-S inclusions, using three different induction methods in SH-SY5Y cells and rat-derived primary neuronal cells. Using confocal and STED microscopy we observed induction-dependent differences in α-S inclusion morphology, location and function. The aggregation of α-S in functionally different compartments correlates with the toxicity of the induction method measured in viability assays. The most cytotoxic treatment largely correlates with the formation of proteasome-associated, juxta-nuclear inclusions. With less toxic methods cytosolic deposits that are not associated with the proteasome are more prevalent. The distribution of α-S over at least two different types of inclusions is not limited to cell models, but is also observed in primary neuronal cells and in human mesencephalon. The existence of functionally different LBs, in vivo and in vitro, gives important insights in the impact of Lewy Body formation on neuronal functioning and may thereby provide a platform for discovering therapeutics.

The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct "Legionella-containing vacuole" (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.