The cellular protrusions for inter-cellular material transfer: similarities between filopodia, cytonemes, tunneling nanotubes, viruses, and extracellular vesicles.

Extracellular vesicles (EVs) are crucial for transferring bioactive materials between cells and play vital roles in both health and diseases. Cellular protrusions, including filopodia and microvilli, are generated by the bending of the plasma membrane and are considered to be rigid structures facilitating various cellular functions, such as cell migration, adhesion, and environment sensing. Compelling evidence suggests that these protrusions are dynamic and flexible structures that can serve as sources of a new class of EVs, highlighting the unique role they play in intercellular material transfer. Cytonemes are specialized filopodia protrusions that make direct contact with neighboring cells, mediating the transfer of bioactive materials between cells through their tips. In some cases, these tips fuse with the plasma membrane of neighboring cells, creating tunneling nanotubes that directly connect the cytosols of the adjacent cells. Additionally, virus particles can be released from infected cells through small bud-like of plasma membrane protrusions. These different types of protrusions, which can transfer bioactive materials, share common protein components, including I-BAR domain-containing proteins, actin cytoskeleton, and their regulatory proteins. The dynamic and flexible nature of these protrusions highlights their importance in cellular communication and material transfer within the body, including development, cancer progression, and other diseases.

Pulmonary Adenocarcinoma with Cutaneous Metastasis in a Dog.

Primary lung cancer is rare in dogs and depending on the tumour stage and subtype, the prognosis can be poor. In this report, we describe a 10 year-old female intact Yorkshire terrier that presented progressive weight loss and chronic pain of unknown origin. Due to the poor condition of the dog, it was subsequently euthanized. Post-mortem evaluation revealed a single large mass in the left caudal lung lobe, with numerous pale, proliferative lesions of various sizes dispersed throughout all the lobes. Additionally, a solitary skin mass was palpated on the mid-thoracic body wall. Histopathological examination of the lung samples revealed multiple distinct, non-encapsulated, expansive neoplastic epithelial cell proliferations with dense cellularity, exhibiting growth patterns, ranging from papillary to micropapillary to solid, accompanied by central areas of necrosis. In some areas, microvilli-like structures were observed on the luminal cytoplasmic margins of the neoplastic cells. The histopathology of the skin mass closely resembled that of the lung. Electron microscopy of the skin samples revealed regions containing cells resembling the respiratory epithelium, along with cells exhibiting processes or microvilli indicative of cilia. The diagnosis was pulmonary adenocarcinoma with cutaneous metastasis. This is the first report of a canine with primary lung cancer that metastasized to the skin.

Induced Formation of Plasma Membrane Protrusions with Porous Materials as Instructive Surfaces.

The plasma membrane is a vital component in cellular processes, and its structure has a significant impact on cellular behavior. The physical characteristics of the extracellular environment, along with the presence of surface pores, can influence the formation of membrane protrusions. Nanoporous surfaces have demonstrated their capacity to induce membrane protrusions in both adherent and non-adherent cells. This chapter presents a methodology that utilizes a nanoporous substrate with nanotopographical constraints to effectively stimulate the formation of membrane protrusions in cells.

Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin.

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Photoreceptor calyceal processes accompany the developing outer segment, adopting a stable length despite a dynamic core.

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.

A quantitative study of the development of taste pores in mice.

OBJECTIVES: This study determined the early development of taste buds by observing the changes in the three-dimensional structures of taste pores and microvilli in the circumvallate papillae (CVP) of mice, from pre- and postnatal stages to the adult stages. METHODS: Fragments of mouse CVP tissue were collected on embryonic day (E) 18 and postnatal days (P) 0, 3, 6, 7, 14, 21, 28, and 56. The surfaces of the tissue fragments located pore apertures via scanning electron microscopy, and the sizes of the CVP and maximum diameters of the pores were estimated from the recorded images. Likewise, changes in the structures of the epithelium around the pore aperture and microvilli protruding from the pores were examined. RESULTS: The size of the CVP exhibited a linear increase with age from E18 to P56. The epithelium around the pore aperture demonstrated changes to form microridges, indicating a characteristic pattern during CVP development. The size of the pore aperture also increased with age from E18 to P56. Furthermore, an increase in the number of pores with protruding microvilli was observed at the base of the epithelial trench. A significant positive correlation was observed between the maximum diameter of the pore and the size of the CVP. CONCLUSIONS: The expansion in the lateral view of the CVP was associated with the developmental stage from E18 to P56, suggesting that the growth of the CVP leads to the opening and enlargement of the taste pores with microvillus projections during these stages.

ARHGAP18-ezrin functions as an autoregulatory module for RhoA in the assembly of distinct actin-based structures.

The location of different actin-based structures is largely regulated by Rho GTPases through specific effectors. We use the apical aspect of epithelial cells as a model system to investigate how RhoA is locally regulated to contribute to two distinct adjacent actin-based structures. Assembly of the non-muscle myosin-2 filaments in the terminal web is dependent on RhoA activity, and assembly of the microvilli also requires active RhoA for phosphorylation and activation of ezrin. We show that the RhoGAP, ARHGAP18, is localized by binding active microvillar ezrin, and this interaction enhances ARHGAP18's RhoGAP activity. We present a model where ezrin-ARHGAP18 acts as a negative autoregulatory module to locally reduce RhoA activity in microvilli. Consistent with this model, loss of ARHGAP18 results in disruption of the distinction between microvilli and the terminal web including aberrant assembly of myosin-2 filaments forming inside microvilli. Thus, ARHGAP18, through its recruitment and activation by ezrin, fine-tunes the local level of RhoA to allow for the appropriate distribution of actin-based structures between the microvilli and terminal web. As RhoGAPs vastly outnumber Rho GTPases, this may represent a general mechanism whereby individual Rho effectors drive specific actin-based structures.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Microvilli regulate the release modes of alpha-tectorin to organize the domain-specific matrix architecture of the tectorial membrane.

The tectorial membrane (TM) is an apical extracellular matrix (ECM) in the cochlea essential for auditory transduction. The TM exhibits highly ordered domain-specific architecture. Alpha-tectorin/TECTA is a glycosylphosphatidylinositol (GPI)-anchored ECM protein essential for TM organization. Here, we identified that TECTA is released by distinct modes: proteolytic shedding by TMPRSS2 and GPI-anchor-dependent release from the microvillus tip. In the medial/limbal domain, proteolytically shed TECTA forms dense fibers. In the lateral/body domain produced by the supporting cells displaying dense microvilli, the proteolytic shedding restricts TECTA to the microvillus tip and compartmentalizes the collagen-binding site. The tip-localized TECTA, in turn, is released in a GPI-anchor-dependent manner to form collagen-crosslinking fibers, required for maintaining the spacing and parallel organization of collagen fibrils. Overall, we showed that distinct release modes of TECTA determine the domain-specific organization pattern, and the microvillus coordinates the release modes along its membrane to organize the higher-order ECM architecture.

PAR-4/LKB1 prevents intestinal hyperplasia by restricting endoderm specification in Caenorhabditis elegans embryos.

The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation.

Pre-organized landscape of T cell surface.

T cell activation is initiated by the recognition of specific antigenic peptides and subsequently accomplished by complex signaling cascades. These aspects have been extensively studied for decades as pivotal factors in the establishment of adaptive immunity. However, how receptors or signaling molecules are organized in the resting state prior to encountering antigens has received less attention. Recent advancements in super-resolution microscopy techniques have revealed topographically controlled pre-formed organization of key molecules involved in antigen recognition and signal transduction on microvillar projections of T cells before activation and substantial effort has been dedicated to characterizing the topological structure of resting T cells over the past decade. This review will summarize our current understanding of how key surface receptors are pre-organized on the T-cell plasma membrane and discuss the potential role of these receptors, which are preassembled prior to ligand binding in the early activation events of T cells.

Dendritic atoh1a+ cells serve as transient intermediates during zebrafish Merkel cell development and regeneration.

Sensory cells often adopt specific morphologies that aid in the detection of external stimuli. Merkel cells encode gentle touch stimuli in vertebrate skin and adopt a reproducible shape characterized by spiky, actin-rich microvilli that emanate from the cell surface. The mechanism by which Merkel cells acquire this stereotyped morphology from basal keratinocyte progenitors is unknown. Here, we establish that dendritic Merkel cells (dMCs) express atonal homolog 1a (atoh1a), extend dynamic filopodial processes, and arise in transient waves during zebrafish skin development and regeneration. We find that dMCs share molecular similarities with both basal keratinocytes and Merkel cells, yet display mesenchymal-like behaviors, including local cell motility and proliferation within the epidermis. Furthermore, dMCs can directly adopt the mature, microvilliated Merkel cell morphology through substantial remodeling of the actin cytoskeleton. Loss of Ectodysplasin A signaling alters the morphology of dMCs and Merkel cells within specific skin regions. Our results show that dMCs represent an intermediate state in the Merkel cell maturation program and identify Ectodysplasin A signaling as a key regulator of Merkel cell morphology.

Defects in microvillus crosslinking sensitize to colitis and inflammatory bowel disease.

Intestinal epithelial cells are covered by the brush border, which consists of densely packed microvilli. The Intermicrovillar Adhesion Complex (IMAC) links the microvilli and is required for proper brush border organization. Whether microvillus crosslinking is involved in the intestinal barrier function or colitis is currently unknown. We investigate the role of microvillus crosslinking in colitis in mice with deletion of the IMAC component CDHR5. Electron microscopy shows pronounced brush border defects in CDHR5-deficient mice. The defects result in severe mucosal damage after exposure to the colitis-inducing agent DSS. DSS increases the permeability of the mucus layer and brings bacteria in direct contact with the disorganized brush border of CDHR5-deficient mice. This correlates with bacterial invasion into the epithelial cell layer which precedes epithelial apoptosis and inflammation. Single-cell RNA sequencing data of patients with ulcerative colitis reveals downregulation of CDHR5 in enterocytes of diseased areas. Our results provide experimental evidence that a combination of microvillus crosslinking defects with increased permeability of the mucus layer sensitizes to inflammatory bowel disease.

Characterization of RACK1-depleted mammalian cells by a palette of microscopy approaches reveals defects in cell cycle progression and polarity establishment.

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.

The Immunosuppressive Potential of Cholesterol Sulfate Through T Cell Microvilli Disruption.

Cholesterol (CL) is required for various biomolecular production processes, including those of cell membrane components. Therefore, to meet these needs, CL is converted into various derivatives. Among these derivatives is cholesterol sulfate (CS), a naturally produced CL derivative by the sulfotransferase family 2B1 (SULT2B1), which is widely present in human plasma. CS is involved in cell membrane stabilization, blood clotting, keratinocyte differentiation, and TCR nanocluster deformation. This study shows that treatment of T cells with CS resulted in the decreased surface expression of some surface T-cell proteins and reduced IL-2 release. Furthermore, T cells treated with CS significantly reduced lipid raft contents and membrane CLs. Surprisingly, using the electron microscope, we also observed that CS led to the disruption of T-cell microvilli, releasing small microvilli particles containing TCRs and other microvillar proteins. However, in vivo, T cells with CS showed aberrant migration to high endothelial venules and limited infiltrating splenic T-cell zones compared with the untreated T cells. Additionally, we observed significant alleviation of atopic dermatitis in mice injected with CS in the animal model. Based on these results, we conclude that CS is an immunosuppressive natural lipid that impairs TCR signaling by disrupting microvillar function in T cells, suggesting its usefulness as a therapeutic agent for alleviating T-cell-mediated hypersensitivity and a potential target for treating autoimmune diseases.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking.

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Microvillar Cartography: A Super-Resolution Single-Molecule Imaging Method to Map the Positions of Membrane Proteins with Respect to Cellular Surface Topography.

We describe microvillar cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, the need for carbon coating, and immunogold clustering, and is amenable to future developments involving, for example, live-cell imaging. This protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell-surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

Dynamics of Immune Cell Microvilli.

Microvilli are actin-based microscopic membrane protrusions that are present in a wide variety of immune cells. Scanning electron microscopy (SEM) revealed that the T cell surface is covered by microvilli. Growing evidence shows that microvilli play important roles in T cell antigen detection and signal transduction. T cell microvilli are highly dynamic and constantly scan and palpate the opposing antigen-presenting cell (APC) surface in search of antigens. Visualizing the rapid movement of microvilli that are only hundreds of nanometers in size requires imaging technologies with high spatial and temporal resolution. Lattice light-sheet microscopy can achieve diffraction-limited resolution in all three dimensions with a temporal resolution of seconds, making it the perfect tool for studying dynamic events of microvilli during T cell antigen detection and activation. In this chapter, we describe a protocol for imaging localization and movement of T cell microvilli and surface receptors using lattice light-sheet microscopy.

T Cell Immunological Synaptosomes: Definition and Isolation.

In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are separated from the T cell body shortly upon TCR stimulation and vesiculated to form T cell microvilli particles (TMPs), a new type of membrane vesicles. TMPs and synaptic ectosomes, which bud from the synaptic cleft, constitute "T cell immunological synaptosomes (TISs)" and act as conveyors of T cell messages or traits to cognate antigen-presenting cells. In practice, it is almost impossible to distinguish between TMPs and synaptic ectosomes. Here, we describe a newly developed protocol to isolate TISs from activated T cells using antibody-immobilized agarose beads and density gradient ultracentrifugation. We further describe the methods for TIS quantification with flow cytometry and to evaluate TIS efficacy on dendritic cells.