Distinct Bin/Amphiphysin/Rvs (BAR) family proteins may assemble on the same tubule to regulate membrane organization in vivo.

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of Drosophila ICA69 (dICA69) (an N-BAR protein) and Drosophila CIP4 (dCIP4) (an F-BAR protein), focusing on their impact on in vivo membrane tubule organization. In contrast to the prevailing models of BAR-domain protein function, we observed colocalization of endogenous dICA69 with dCIP4-induced tubules, indicating their potential recruitment for tubule formation and maintenance. Moreover, actin-regulatory proteins such as Wasp, SCAR, and Arp2/3 were recruited at the site of CIP4-induced tubule formation. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width, possibly by recruiting actin modulators during the generation of tubules. Our data suggests that cooperative activities of distinct BAR-domain family proteins may determine the length and width of the membrane tubule in vivo.

Dynamics of membrane tubulation coupled with fission by a two-component module.

Membrane tubulation coupled with fission (MTCF) is a widespread phenomenon but mechanisms for their coordination remain unclear, partly because of the lack of assays to monitor dynamics of membrane tubulation and subsequent fission. Using polymer cushioned bilayer islands, we analyze the membrane tubulator Bridging Integrator 1 (BIN1) mixed with the fission catalyst dynamin2 (Dyn2). Our results reveal this mixture to constitute a minimal two-component module that demonstrates MTCF. MTCF is an emergent property and arises because BIN1 facilitates recruitment but inhibits membrane binding of Dyn2 in a dose-dependent manner. MTCF is therefore apparent only at high Dyn2 to BIN1 ratios. Because of their mutual involvement in T-tubules biogenesis, mutations in BIN1 and Dyn2 are associated with centronuclear myopathies and our analysis links the pathology with aberrant MTCF. Together, our results establish cushioned bilayer islands as a facile template for the analysis of membrane tubulation and inform of mechanisms that coordinate MTCF.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Amphiphysin-2 (BIN1) functions and defects in cardiac and skeletal muscle.

Amphiphysin-2 is a ubiquitously expressed protein also known as bridging integrator 1 (BIN1), playing a critical role in membrane remodeling, trafficking, and cytoskeleton dynamics in a wide range of tissues. Mutations in the gene encoding BIN1 cause centronuclear myopathies (CNM), and recent evidence has implicated BIN1 in heart failure, underlining its crucial role in both skeletal and cardiac muscle. Furthermore, altered expression of BIN1 is linked to an increased risk of late-onset Alzheimer's disease and several types of cancer, including breast, colon, prostate, and lung cancers. Recently, the first proof-of-concept for potential therapeutic strategies modulating BIN1 were obtained for muscle diseases. In this review article, we discuss the similarities and differences in BIN1's functions in cardiac and skeletal muscle, along with its associated diseases and potential therapies.

Recognition and remodeling of endosomal zones by sorting nexins.

The proteolipid code determines how cytosolic proteins find and remodel membrane surfaces. Here, we investigate how this process works with sorting nexins Snx1 and Snx3. Both proteins form sorting machines by recognizing membrane zones enriched in phosphatidylinositol 3-phosphate (PI3P), phosphatidylserine (PS) and cholesterol. This co-localized combination forms a unique "lipid codon" or lipidon that we propose is responsible for endosomal targeting, as revealed by structures and interactions of their PX domain-based readers. We outline a membrane recognition and remodeling mechanism for Snx1 and Snx3 involving this code element alongside transmembrane pH gradients, dipole moment-guided docking and specific protein-protein interactions. This generates an initial membrane-protein assembly (memtein) that then recruits retromer and additional PX proteins to recruit cell surface receptors for sorting to the trans-Golgi network (TGN), lysosome and plasma membranes. Post-translational modification (PTM) networks appear to regulate how the sorting machines form and operate at each level. The commonalities and differences between these sorting nexins show how the proteolipid code orchestrates parallel flows of molecular information from ribosome emergence to organelle genesis, and illuminates a universally applicable model of the membrane.

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation.

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

EHBP1 Is Critically Involved in the Dendritic Arbor Formation and Is Coupled to Factors Promoting Actin Filament Formation.

The coordinated action of a plethora of factors is required for the organization and dynamics of membranous structures critically underlying the development and function of cells, organs, and organisms. The evolutionary acquisition of additional amino acid motifs allows for expansion and/or specification of protein functions. We identify a thus far unrecognized motif specific for chordata EHBP1 proteins and demonstrate that this motif is critically required for interaction with syndapin I, an F-BAR domain-containing, membrane-shaping protein predominantly expressed in neurons. Gain-of-function and loss-of-function studies in rat primary hippocampal neurons (of mixed sexes) unraveled that EHBP1 has an important role in neuromorphogenesis. Surprisingly, our analyses uncovered that this newly identified function of EHBP1 did not require the domain responsible for Rab GTPase binding but was strictly dependent on EHBP1's syndapin I binding interface and on the presence of syndapin I in the developing neurons. These findings were underscored by temporally and spatially remarkable overlapping dynamics of EHBP1 and syndapin I at nascent dendritic branch sites. In addition, rescue experiments demonstrated the necessity of two additional EHBP1 domains for dendritic arborization, the C2 and CH domains. Importantly, the additionally uncovered critical involvement of the actin nucleator Cobl in EHBP1 functions suggested that not only static association with F-actin via EHBP1's CH domain is important for dendritic arbor formation but also actin nucleation. Syndapin interactions organize ternary protein complexes composed of EHBP1, syndapin I, and Cobl, and our functional data show that only together these factors give rise to proper cell shape during neuronal development.

MTSS1 is downregulated in nasopharyngeal carcinoma (NPC) which disrupts adherens junctions leading to enhanced cell migration and invasion.

Loss of cell-cell adhesions is the indispensable first step for cancer cells to depart from the primary tumor mass to metastasize. Metastasis suppressor 1 (MTSS1) is frequently lost in metastatic tissues, correlating to advanced tumor stages and poor prognosis across a variety of cancers. Here we explore the anti-metastatic mechanisms of MTSS1, which have not been well understood. We found that MTSS1 is downregulated in NPC tissues. Lower levels of MTSS1 expression correlate to worse prognosis. We show that MTSS1 suppresses NPC cell migration and invasion in vitro through cytoskeletal remodeling at cell-cell borders and assembly of E-cadherin/ß-catenin/F-actin in adherens junctions. The I-BAR domain of MTSS1 was both necessary and sufficient to restore this formation of E-cadherin/ß-catenin/F-actin-mediated cell adherens junctions.

MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis.

Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis.

Tetraspanner-based nanodomains modulate BAR domain-induced membrane curvature.

The topography of biological membranes is critical for formation of protein and lipid microdomains. One prominent example in the yeast plasma membrane (PM) are BAR domain-induced PM furrows. Here we report a novel function for the Sur7 family of tetraspanner proteins in the regulation of local PM topography. Combining TIRF imaging, STED nanoscopy, freeze-fracture EM and membrane simulations we find that Sur7 tetraspanners form multimeric strands at the edges of PM furrows, where they modulate forces exerted by BAR domain proteins at the furrow base. Loss of Sur7 tetraspanners or Sur7 displacement due to altered PIP2 homeostasis leads to increased PM invagination and a distinct form of membrane tubulation. Physiological defects associated with PM tubulation are rescued by synthetic anchoring of Sur7 to furrows. Our findings suggest a key role for tetraspanner proteins in sculpting local membrane domains. The maintenance of stable PM furrows depends on a balance between negative curvature at the base which is generated by BAR domains and positive curvature at the furrows' edges which is stabilized by strands of Sur7 tetraspanners.

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

In Silico Identification and Analysis of Proteins Containing the Phox Homology Phosphoinositide-Binding Domain in Kinetoplastea Protists: Evolutionary Conservation and Uniqueness of Phox-Homology-Domain-Containing Protein Architectures.

Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam's pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs.

Investigating the Mechanical Properties and Flexibility of N-BAR Domains in PICK1 by Molecular Dynamics Simulations.

INTRODUCTION: The proteins of the Bin/Amphiphysin/Rvs167 (BAR) domain superfamily are believed to induce membrane curvature. PICK1 is a distinctive protein that consists of both a BAR and a PDZ domain, and it has been associated with numerous diseases. It is known to facilitate membrane curvature during receptor-mediated endocytosis. In addition to understanding how the BAR domain facilitates membrane curvature, it's particularly interesting to unravel the hidden links between the structural and mechanical properties of the PICK1 BAR domain. METHODS: This paper employs steered molecular dynamics (SMD) to investigate the mechanical properties associated with structural changes in the PICK1 BAR domains. RESULTS: Our findings suggest that not only do helix kinks assist in generating curvature of BAR domains, but they may also provide the additional flexibility required to initiate the binding between BAR domains and the membrane. CONCLUSION: We have observed a complex interaction network within the BAR monomer and at the binding interface of the two BAR monomers. This network is crucial for maintaining the mechanical properties of the BAR dimer. Owing to this interaction network, the PICK1 BAR dimer exhibits different responses to external forces applied in opposite directions.

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Mechanical regulation of synapse formation and plasticity.

Dendritic spines are small protrusions arising from dendrites and constitute the major compartment of excitatory post-synapses. They change in number, shape, and size throughout life; these changes are thought to be associated with formation and reorganization of neuronal networks underlying learning and memory. As spines in the brain are surrounded by the microenvironment including neighboring cells and the extracellular matrix, their protrusion requires generation of force to push against these structures. In turn, neighboring cells receive force from protruding spines. Recent studies have identified BAR-domain proteins as being involved in membrane deformation to initiate spine formation. In addition, forces for dendritic filopodium extension and activity-induced spine expansion are generated through cooperation between actin polymerization and clutch coupling. On the other hand, force from expanding spines affects neurotransmitter release from presynaptic terminals. Here, we review recent advances in our understanding of the physical aspects of synapse formation and plasticity, mainly focusing on spine dynamics.

CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures.

BAR (Bin, Amphiphysin, and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse BAR) domain containing tools derived from the fusion of the Arabidopsis thaliana cryptochrome 2 photoreceptor and I-BAR protein domains ("CRY-BARs") with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane-binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and phosphatidylinositol 4,5-bisphosphate-binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

Role of Bin-Amphiphysin-Rvs (BAR) domain proteins in mediating neuronal signaling and disease.

Several proteins contain signaling domains that can regulate the cell membrane dynamics as well as the underlying cytoskeleton. Among these, Bin-Amphiphysin-Rvs (BAR) domain-containing proteins, with their membrane deforming properties, have emerged as the key players in regulating neuronal morphology and inducing neuronal signaling that can modulate synaptic architecture. While the biochemical and structural basis of membrane deformation by the BAR-domain proteins has been extensively studied, the in vivo contexts in which these proteins function remain to be elucidated. Despite the discovery of BAR-domain proteins over 25 years ago, most of the studies have primarily focused on understanding the structural and biochemical properties and cell biological processes regulated by these proteins. Understanding the functional requirements of these proteins at the level of multicellular organisms and the way these proteins regulate biological processes remains a topic of intensive study. In this review, we discuss the functional roles of BAR-domain proteins in the context of membrane dynamics and cellular signaling. We highlight recent developments describing the functional role of these proteins in neuronal morphogenesis, synaptic function, and disease.

Ca 2+ Regulates Dimerization of the BAR Domain Protein PICK1 and Consequent Membrane Curvature.

Bin-Amphiphysin-Rvs (BAR) domain proteins are critical regulators of membrane geometry. They induce and stabilize membrane curvature for processes, such as clathrin-coated pit formation and endosomal membrane tubulation. BAR domains form their characteristic crescent-shaped structure in the dimeric form, indicating that the formation of the dimer is critical to their function of inducing membrane curvature and suggesting that a dynamic monomer-dimer equilibrium regulated by cellular signaling would be a powerful mechanism for controlling BAR domain protein function. However, to the best of our knowledge, cellular mechanisms for regulating BAR domain dimerization remain unexplored. PICK1 is a Ca2+-binding BAR domain protein involved in the endocytosis and endosomal recycling of neuronal AMPA receptors and other transmembrane proteins. In this study, we demonstrated that PICK1 dimerization is regulated by a direct effect of Ca2+ ions via acidic regions in the BAR domain and at the N-terminus. While the cellular membrane tubulating activity of PICK1 is absent under basal conditions, Ca2+ influx causes the generation of membrane tubules that originate from the cell surface. Furthermore, in neurons, PICK1 dimerization increases transiently following NMDA receptor stimulation. We believe that this novel mechanism for regulating BAR domain dimerization and function represents a significant conceptual advance in our knowledge about the regulation of cellular membrane curvature.

Time-Resolved Fluorescence Microscopy Screens on Host Protein Subversion During Bacterial Cell Invasion.

Intracellular bacterial pathogens have evolved a plethora of strategies to invade eukaryotic cells. By manipulating host signaling pathways, in particular vesicular trafficking, these microbes subvert host functions to promote their internalization and to establish an intracellular niche. During these events, host endomembrane compartments are dynamically reorganized. Shigella flexneri, the causative agent of bacillary dysentery, recruits components of the host recycling pathway and the exocyst of non-phagocytic enterocytes in the vicinity of its entry site to facilitate its access to the host cytosol. These factors are either dynamically tethered to in situ formed macropinosomes or to the bacteria-containing vacuole itself. The underlying interactions cannot readily be monitored as individual bacterial infection events take place without synchronicity using cellular infection models. Therefore, time-resolved screens by fluorescence microscopy represent a powerful tool for the study of host subversion. Such screens can be performed with libraries of fluorescently tagged host factors. Using the cytosolic pathogenic agent Shigella flexneri as a model, we provide detailed protocols for such medium-to-high throughput multidimensional imaging screening of the dynamic host-pathogen cross talk. Our workflow is designed to be easily adapted for the study of different host factor libraries and different pathogen models.