Pericyte dysfunction and loss of interpericyte tunneling nanotubes promote neurovascular deficits in glaucoma.

Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.

Connecting different heart diseases through intercellular communication.

Well-orchestrated intercellular communication networks are pivotal to maintaining cardiac homeostasis and to ensuring adaptative responses and repair after injury. Intracardiac communication is sustained by cell-cell crosstalk, directly via gap junctions (GJ) and tunneling nanotubes (TNT), indirectly through the exchange of soluble factors and extracellular vesicles (EV), and by cell-extracellular matrix (ECM) interactions. GJ-mediated communication between cardiomyocytes and with other cardiac cell types enables electrical impulse propagation, required to sustain synchronized heart beating. In addition, TNT-mediated organelle transfer has been associated with cardioprotection, whilst communication via EV plays diverse pathophysiological roles, being implicated in angiogenesis, inflammation and fibrosis. Connecting various cell populations, the ECM plays important functions not only in maintaining the heart structure, but also acting as a signal transducer for intercellular crosstalk. Although with distinct etiologies and clinical manifestations, intercellular communication derailment has been implicated in several cardiac disorders, including myocardial infarction and hypertrophy, highlighting the importance of a comprehensive and integrated view of complex cell communication networks. In this review, I intend to provide a critical perspective about the main mechanisms contributing to regulate cellular crosstalk in the heart, which may be considered in the development of future therapeutic strategies, using cell-based therapies as a paradigmatic example. This Review has an associated Future Leader to Watch interview with the author.

Membrane nanotubes are ancient machinery for cell-to-cell communication and transport. Their interference with the immune system.

Nanotubular connections between mammalian cell types came into the focus only two decades ago, when "live cell super-resolution imaging" was introduced. Observations of these long-time overlooked structures led to understanding mechanisms of their growth/withdrawal and exploring some key genetic and signaling factors behind their formation. Unbelievable level of multiple supportive collaboration between tumor cells undergoing cytotoxic chemotherapy, cross-feeding" between independent bacterial strains or "cross-dressing" collaboration of immune cells promoting cellular immune response, all via nanotubes, have been explored recently. Key factors and "calling signals" determining the spatial directionality of their growth and their overall in vivo significance, however, still remained debated. Interestingly, prokaryotes, including even ancient archaebacteria, also seem to use such NT connections for intercellular communication. Herein, we will give a brief overview of current knowledge of membrane nanotubes and depict a simple model about their possible "historical role".

Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-ß aggregates.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aß) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aß/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear. METHODS: The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aß pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aß and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space. RESULTS: Our data show that intracellular deposits of αSYN and Aß are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte. CONCLUSIONS: Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aß/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.

Cytomembrane visualization using Stokes parameter confocal microscopy.

A new, to the best of our knowledge, method for Stokes vector imaging is proposed to achieve imaging and dynamic monitoring of a non-labeled cytomembrane. In this work, a polarization state vector is described by a Stokes vector and expressed in chrominance space. A physical quantity called polarization chromaticity value (PCV) corresponding to a Stokes vector is used as the imaging parameter to perform Stokes vector imaging. By using the PCV imaging technique, the Stokes vector can be expressed in three-dimensional real space rather than in a Poincare sphere. Furthermore, a four-way Stokes parameter confocal microscopy system is designed to measure four Stokes parameters simultaneously and obtain micro-imaging. Label-free living onion cell membranes and their plasmolysis process are selected as the representative micro-anisotropy experimental analysis. It is proved that PCV imaging can perform visualization of cytomembranes, and further, microscopic orientation is demonstrated. The prospect of universal measurement of anisotropy details for analysis and diagnosis is provided.

RNA transfer through tunneling nanotubes.

It was already suggested in the early '70's that RNA molecules might transfer between mammalian cells in culture. Yet, more direct evidence for RNA transfer in animal and plant cells was only provided decades later, as this field became established. In this mini-review, we will describe evidence for the transfer of different types of RNA between cells through tunneling nanotubes (TNTs). TNTs are long, yet thin, open-ended cellular protrusions that are structurally distinct from filopodia. TNTs connect cells and can transfer many types of cargo, including small molecules, proteins, vesicles, pathogens, and organelles. Recent work has shown that TNTs can also transfer mRNAs, viral RNAs and non-coding RNAs. Here, we will review the evidence for TNT-mediated RNA transfer, discuss the technical challenges in this field, and conjecture about the possible significance of this pathway in health and disease.

Kinematics Governing Mechanotransduction in the Sensory Hair of the Venus flytrap.

Insects fall prey to the Venus flytrap (Dionaea muscipula) when they touch the sensory hairs located on the flytrap lobes, causing sudden trap closure. The mechanical stimulus imparted by the touch produces an electrical response in the sensory cells of the trigger hair. These cells are found in a constriction near the hair base, where a notch appears around the hair's periphery. There are mechanosensitive ion channels (MSCs) in the sensory cells that open due to a change in membrane tension; however, the kinematics behind this process is unclear. In this study, we investigate how the stimulus acts on the sensory cells by building a multi-scale hair model, using morphometric data obtained from µ-CT scans. We simulated a single-touch stimulus and evaluated the resulting cell wall stretch. Interestingly, the model showed that high stretch values are diverted away from the notch periphery and, instead, localized in the interior regions of the cell wall. We repeated our simulations for different cell shape variants to elucidate how the morphology influences the location of these high-stretch regions. Our results suggest that there is likely a higher mechanotransduction activity in these 'hotspots', which may provide new insights into the arrangement and functioning of MSCs in the flytrap.

A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels.

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.

Protease-resistant cell meshworks: An indication of membrane nanotube-based syncytia formation.

Cell biology considers most animal tissues as assemblies of "individual" cells that rely on different contact-dependent communication mechanisms, including synapses, gap junctions or - a recent awareness - membrane nano- and microtubes. However, by protease-mediated singularization of dense 2D/ 3D cell cultures and tissue explants, we show here that cell collectives stay connected via a continuous meshwork of F-actin-based membrane tubes, resembling tunneling nanotube (TNT)-based networks observed between dispersed cell cultures. Fusion of respective tubes was accompanied by the ingrowth of microtubules and the invasion of mitochondria and lysosomes. Remarkably, in homology to the plasmodesmata-based plant symplast, we found evidence for expanded, membrane-based syncytia in animal tissues by observing dye transfer among the highly interlinked cells. This approach allows for the first time to visualize and quantify membrane continuity-based connections among densely packed cells and to assess their potential physiological and pathological impact closer to the in vivo situation.

Eisosomes.

Moseley discusses the molecular and mechanical functions of eisosomes - invaginations from the yeast plasma membrane.

Caveolae.

Caveolae are one of the most abundant and striking features of the plasma membrane of many mammalian cell types. These surface pits have fascinated biologists since their discovery by the pioneers of electron microscopy in the middle of the last century, but we are only just starting to understand their multiple functions. Molecular understanding of caveolar formation is advancing rapidly and we now know that sculpting the membrane to generate the characteristic bulb-shaped caveolar pit involves the coordinated action of integral membrane proteins and peripheral membrane coat proteins in a process dependent on their multiple interactions with membrane lipids. The resulting structure is further stabilised by protein complexes at the caveolar neck. Caveolae can bud to generate an endocytic carrier but can also be disassembled in response to specific stimuli to function as a mechanoprotective device. These structures have also been linked to numerous signalling pathways. Here, we will briefly summarise the current molecular and structural understanding of caveolar formation and dynamics, discuss how the crucial structural components of caveolae work together to generate a dynamic sensing domain, and discuss the implications of recent studies on the diverse roles proposed for caveolae in different cells and tissues.

Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t-tubule membranes in mouse ventricular myocytes.

Cholesterol is a key component of the cell plasma membrane. It has been suggested that the t-tubule membrane of cardiac ventricular myocytes is enriched in cholesterol and that this plays a role in determining t-tubule structure and function. We have used methyl-ß-cyclodextrin (MßCD) to deplete cholesterol in intact and detubulated mouse ventricular myocytes to investigate the contribution of cholesterol to t-tubule structure, membrane capacitance, and the distribution of Ca flux pathways. Depletion of membrane cholesterol was confirmed using filipin; however, di-8-ANEPPS staining showed no differences in t-tubule structure following MßCD treatment. MßCD treatment had no significant effect on the capacitance:volume relationship of intact myocytes or on the decrease in capacitance:volume caused by detubulation. Similarly, Ca influx and efflux were not altered by MßCD treatment and were reduced by a similar amount following detubulation in untreated and MßCD-treated cells. These data show that cholesterol depletion has similar effects on the surface and t-tubule membranes and suggest that cholesterol plays no acute role in determining t-tubule structure and function.

Regulation of circular dorsal ruffles, macropinocytosis, and cell migration by RhoG and its exchange factor, Trio.

Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases. So far, only Rac1 has been consistently associated with CDR formation, whereas the role of other GTPases in this process is either lacking or inconclusive. Here we show that RhoG and its exchange factor, Trio, play a role in the regulation of CDR dynamics, particularly by modulating their size. RhoG is activated by Trio downstream of PDGF in a PI3K- and Src-dependent manner. Silencing RhoG expression decreases the number of cells that form CDRs, as well as the area of the CDRs. The regulation of CDR area by RhoG is independent of Rac1 function. In addition, our results show the RhoG plays a role in the cellular functions associated with CDR formation, including macropinocytosis, receptor internalization, and cell migration. Taken together, our results reveal a novel role for RhoG in the regulation of CDRs and the cellular processes associated with their formation.

A new mechanism for spatial pattern formation via lateral and protrusion-mediated lateral signalling.

Tissue organization and patterning are critical during development when genetically identical cells take on different fates. Lateral signalling plays an important role in this process by helping to generate self-organized spatial patterns in an otherwise uniform collection of cells. Recent data suggest that lateral signalling can be mediated both by junctional contacts between neighbouring cells and via cellular protrusions that allow non-neighbouring cells to interact with one another at a distance. However, it remains unclear precisely how signalling mediated by these distinct types of cell-cell contact can physically contribute to the generation of complex patterns without the assistance of diffusible morphogens or pre-patterns. To explore this question, in this work we develop a model of lateral signalling based on a single receptor/ligand pair as exemplified by Notch and Delta. We show that allowing the signalling kinetics to differ at junctional versus protrusion-mediated contacts, an assumption inspired by recent data which show that the cleavage of Notch in several systems requires both Delta binding and the application of mechanical force, permits individual cells to act to promote both lateral activation and lateral inhibition. Strikingly, under this model, in which Delta can sequester Notch, a variety of patterns resembling those typical of reaction-diffusion systems is observed, together with more unusual patterns that arise when we consider changes in signalling kinetics, and in the length and distribution of protrusions. Importantly, these patterns are self-organizing-so that local interactions drive tissue-scale patterning. Together, these data show that protrusions can, in principle, generate different types of patterns in addition to contributing to long-range signalling and to pattern refinement.

Leading from the Back: The Role of the Uropod in Neutrophil Polarization and Migration.

Cell motility is required for diverse biological processes including development, homing of immune cells, wound healing, and cancer cell invasion. Motile neutrophils exhibit a polarized morphology characterized by the formation of leading-edge pseudopods and a highly contractile cell rear known as the uropod. Although it is known that perturbing uropod formation impairs neutrophil migration, the role of the uropod in cell polarization and motility remains incompletely understood. Here we discuss cell intrinsic mechanisms that regulate neutrophil polarization and motility, with a focus on the uropod, and examine how relationships among regulatory mechanisms change when cells change their direction of migration.

How Our Other Genome Controls Our Epi-Genome.

Eukaryotes and prokaryotes produce extracellular nanovescicles that contain RNAs and other molecules that they exploit to communicate. Recently, inter-kingdom crosstalk was demonstrated between humans and bacteria through fecal microRNAs. We suggest here how bacteria interact with humans via RNAs within membrane vesicles to alter our epigenome, thus filling the gap and closing the circle. At the same time, there are indications that there could be a wider inter-kingdom communication network that might encompass all known kingdoms. Now that the connection with our other genome has been established, we also should begin to explore the 'social' network that we have around us.

Higher-order assemblies of BAR domain proteins for shaping membranes.

Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions.

Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

StARTing to understand membrane contact sites.

Lipid transport proteins localized to membrane contact sites mediate the distribution of lipids between organelles. Recently identified StART-like sterol transporting proteins in yeast can explain sterol delivery within the cell. The multiple localizations of these proteins could provide alternative routes for sterol delivery and mediate inter-regulation of membrane contact sites.