Ceramide-mediated orchestration of oxidative stress response through filopodia-derived small extracellular vesicles.

Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H2O2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150 nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia-derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopodia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress-induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S-palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD-ceramide. NBD-ceramide-labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclusion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide-rich sEVs that target mitochondria and propagate cell death.

Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy.

Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins.

Filopodia: integrating cellular functions with theoretical models.

Filopodia, widely distributed on cell surfaces, are distinguished by their dynamic extensions, playing pivotal roles in a myriad of biological processes. Their functions span from mechanosensing and guidance to cell-cell communication during cellular organization in the early embryo. Filopodia have significant roles in pathogenic processes, such as cancer invasion and viral dissemination. Molecular mapping of the filopodome has revealed generic components essential for filopodia functions. In parallel, recent insights into biophysical mechanisms governing filopodia dynamics have provided the foundation for broader investigations of filopodia's biological functions. We highlight recent discoveries of engagement of filopodia in various stages of development and pathogenesis and present an overview of intricate molecular and physical features of these cellular structures across a spectrum of cellular activities.

Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology Dynamics.

Super-resolution fluorescence imaging has emerged as a potent tool for investigating the nanoscale structure and function of the plasma membrane (PM). Nevertheless, the challenge persists in achieving super-resolution imaging of PM dynamics due to limitations in probe photostability and issues with cell internalization staining. Herein, we report assembly-mediated buffering fluorogenic probes BMP-14 and BMP-16 exhibiting fast PM labeling and extended retention time (over 2 h) on PM. The incorporation of alkyl chains proves effective in promoting the aggregation of BMP-14 and BMP-16 into nonfluorescent nanoparticles to realize fluorogenicity and regulate the buffering capacity to rapidly replace photobleached probes ensuring stable long-term super-resolution imaging of PM. Utilizing these PM-buffering probes, we observed dynamic movements of PM filopodia and continuous shrinkage, leading to the formation of extracellular vesicles (EVs) using structured illumination microscopy (SIM). Furthermore, we discovered two distinct modes of EV fusion: one involving fusion through adjacent lipids and the other through filamentous lipid traction. The entire process of EV fusion outside the PM was dynamically tracked. Additionally, BMP-16 exhibited a unique capability of inducing single-molecule fluorescence blinking when used for cell membrane staining. This property makes BMP-16 suitable for the PAINT imaging of cell membranes.

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.

SOX4 induces cytoskeleton remodeling and promotes cell motility via N-wasp/ARP2/3 pathway in colorectal cancer cells.

Filopodia are thin, actin-rich projection from the plasma membrane that promote cancer cell invasion and migration. Sex-determining region Y-related high-mobility group-box 4 (SOX4) is a crucial transcription factor that plays a role in the development and metastasis of colorectal cancer (CRC). However, the involvement of SOX4 in cytoskeleton remodeling in CRC remains unknown. For the first time, we demonstrate that SOX4 is a potent regulator of filopodia formation in CRC cells. Overexpression of SOX4 protein enhances both migration and invasion ability of HCT116, and CACO2 cells, which is relevant to the metastasis. Furthermore, through phalloidin staining, cytoskeleton re-assembly was observed in SOX4-modified cell lines. Enhanced expression of SOX4 increased the number and length of filopodia on cell surface. In contrast, silencing SOX4 in SW620 cells with higher endogenous expression of SOX4, impeded the filopodia formation. Moreover, SOX4 was found to be positively regulating the expression of central regulators of actin cytoskeleton - N-Wiskott-Aldrich syndrome protein (N-WASP); WAVE2; Actin related proteins, ARP2 and ARP3. Inhibiting the N-WASP/ARP2/3 pathway diminishes the filopodia formation and the migration of CRC cells. These results indicate the crucial role of SOX4 in the regulation of filopodia formation mediated by N-WASP/ARP2/3 pathway in CRC cells.

ECL cytosensor for sensitive and label-free detection of circulating tumor cells based on hierarchical flower-like gold microstructures.

The development of sensitive and efficient cell sensing strategies to detect circulating tumor cells (CTCs) in peripheral blood is crucial for the early diagnosis and prognostic assessment of cancer clinical treatment. Herein, an array of hierarchical flower-like gold microstructures (HFGMs) with anisotropic nanotips was synthesized by a simple electrodeposition method and used as a capture substrate to construct an ECL cytosensor based on the specific recognition of target cells by aptamers. The complex topography of the HFGMs array not only catalyzed the enhancement of ECL signals, but also induced the cells to generate more filopodia, improving the capture efficiency and shortening the capture time. The effect of topographic roughness on cell growth and adhesion propensity was also investigated, while the cell capture efficiency was proposed to be an important indicator affecting the accuracy of the ECL cytosensor. In addition, the capture of cells on the electrode surface increased the steric hindrance, which caused ECL signal changes in the Ru(bpy)32+ and TPrA system, realizing the quantitative detection of MCF-7 cells. The detection range of the sensor was from 102 to 106 cells mL-1 and the detection limit was 18 cells mL-1. The proposed detection method avoids the process of separation, labeling and counting, which has great potential for sensitive detection in clinical applications.

Oleate Promotes Triple-Negative Breast Cancer Cell Migration by Enhancing Filopodia Formation through a PLD/Cdc42-Dependent Pathway.

Breast cancer, particularly triple-negative breast cancer (TNBC), poses a global health challenge. Emerging evidence has established a positive association between elevated levels of stearoyl-CoA desaturase 1 (SCD1) and its product oleate (OA) with cancer development and metastasis. SCD1/OA leads to alterations in migration speed, direction, and cell morphology in TNBC cells, yet the underlying molecular mechanisms remain elusive. To address this gap, we aim to investigate the impact of OA on remodeling the actin structure in TNBC cell lines, and the underlying signaling. Using TNBC cell lines and bioinformatics tools, we show that OA stimulation induces rapid cell membrane ruffling and enhances filopodia formation. OA treatment triggers the subcellular translocation of Arp2/3 complex and Cdc42. Inhibiting Cdc42, not the Arp2/3 complex, effectively abolishes OA-induced filopodia formation and cell migration. Additionally, our findings suggest that phospholipase D is involved in Cdc42-dependent filopodia formation and cell migration. Lastly, the elevated expression of Cdc42 in breast tumor tissues is associated with a lower survival rate in TNBC patients. Our study outlines a new signaling pathway in the OA-induced migration of TNBC cells, via the promotion of Cdc42-dependent filopodia formation, providing a novel insight for therapeutic strategies in TNBC treatment.

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

Unlocking mechanosensitivity: integrins in neural adaptation.

Mechanosensitivity extends beyond sensory cells to encompass most neurons in the brain. Here, we explore recent research on the role of integrins, a diverse family of adhesion molecules, as crucial biomechanical sensors translating mechanical forces into biochemical and electrical signals in the brain. The varied biomechanical properties of neuronal integrins, including their force-dependent conformational states and ligand interactions, dictate their specific functions. We discuss new findings on how integrins regulate filopodia and dendritic spines, shedding light on their contributions to synaptic plasticity, and explore recent discoveries on how they engage with metabotropic receptors and ion channels, highlighting their direct participation in electromechanical transduction. Finally, to facilitate a deeper understanding of these developments, we present molecular and biophysical models of mechanotransduction.

Transport and gradient formation of Wnt and Fgf in the early zebrafish gastrula.

Within embryonic development, the occurrence of gastrulation is critical in the formation of multiple germ layers with many differentiative abilities. These cells are instructed through exposure to signalling molecules called morphogens. The secretion of morphogens from a source tissue creates a concentration gradient that allows distinct pattern formation in the receiving tissue. This review focuses on the morphogens Wnt and Fgf in zebrafish development. Wnt has been shown to have critical roles throughout gastrulation, including in anteroposterior patterning and neural posterisation. Fgf is also a vital signal, contributing to involution and mesodermal specification. Both morphogens have also been found to work in finely balanced synergy for processes such as neural induction. Thus, the signalling range of Wnts and Fgfs must be strictly controlled to target the correct target cells. Fgf and Wnts signal to local cells as well as to cells in the distance in a highly regulated way, requiring specific dissemination mechanisms that allow efficient and precise signalling over short and long distances. Multiple transportation mechanisms have been discovered to aid in producing a stable morphogen gradient, including short-range diffusion, filopodia-like extensions called cytonemes and extracellular vesicles, mainly exosomes. These mechanisms are specific to the morphogen that they transport and the intended signalling range. This review article discusses how spreading mechanisms in these two morphogenetic systems differ and the consequences on paracrine signalling, hence tissue patterning.

KAT8/SIRT7-mediated Fascin-K41 acetylation/deacetylation regulates tumor metastasis in esophageal squamous cell carcinoma.

Fascin actin-bundling protein 1 (Fascin) is highly expressed in a variety of cancers, including esophageal squamous cell carcinoma (ESCC), working as an important oncogenic protein and promoting the migration and invasion of cancer cells by bundling F-actin to facilitate the formation of filopodia and invadopodia. However, it is not clear how exactly the function of Fascin is regulated by acetylation in cancer cells. Here, in ESCC cells, the histone acetyltransferase KAT8 catalyzed Fascin lysine 41 (K41) acetylation, to inhibit Fascin-mediated F-actin bundling and the formation of filopodia and invadopodia. Furthermore, NAD-dependent protein deacetylase sirtuin (SIRT) 7-mediated deacetylation of Fascin-K41 enhances the formation of filopodia and invadopodia, which promotes the migration and invasion of ESCC cells. Clinically, the analysis of cancer and adjacent tissue samples from patients with ESCC showed that Fascin-K41 acetylation was lower in the cancer tissue of patients with lymph node metastasis than in that of patients without lymph node metastasis, and low levels of Fascin-K41 acetylation were associated with a poorer prognosis in patients with ESCC. Importantly, K41 acetylation significantly blocked NP-G2-044, one of the Fascin inhibitors currently being clinically evaluated, suggesting that NP-G2-044 may be more suitable for patients with low levels of Fascin-K41 acetylation, but not suitable for patients with high levels of Fascin-K41 acetylation. © 2024 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

filoVision - using deep learning and tip markers to automate filopodia analysis.

Filopodia are slender, actin-filled membrane projections used by various cell types for environment exploration. Analyzing filopodia often involves visualizing them using actin, filopodia tip or membrane markers. Due to the diversity of cell types that extend filopodia, from amoeboid to mammalian, it can be challenging for some to find a reliable filopodia analysis workflow suited for their cell type and preferred visualization method. The lack of an automated workflow capable of analyzing amoeboid filopodia with only a filopodia tip label prompted the development of filoVision. filoVision is an adaptable deep learning platform featuring the tools filoTips and filoSkeleton. filoTips labels filopodia tips and the cytosol using a single tip marker, allowing information extraction without actin or membrane markers. In contrast, filoSkeleton combines tip marker signals with actin labeling for a more comprehensive analysis of filopodia shafts in addition to tip protein analysis. The ZeroCostDL4Mic deep learning framework facilitates accessibility and customization for different datasets and cell types, making filoVision a flexible tool for automated analysis of tip-marked filopodia across various cell types and user data.

Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

N6-Methyladenosine Modification of lncCCKAR-5 Regulates Autophagy in Human Umbilical Cord Mesenchymal Stem Cells by Destabilizing LMNA and Inhibits Diabetic Wound Healing.

Long noncoding RNAs are pivotal contributors to the development of human diseases. However, their significance in the context of diabetic wound healing regulated by human umbilical cord mesenchymal stem cells (hUCMSCs) remains unclear. This study sheds light on the involvement of lncCCKAR5 in this process. We found that hUCMSCs exposed to high glucose conditions exhibited a significant downregulation of lncCCKAR5 expression, and lncCCKAR5 played a critical role in modulating autophagy, thus inhibiting apoptosis in hUCMSCs. In addition, the reduction of lncCCKAR5 in cells exposed to high glucose effectively thwarted cellular senescence and facilitated filopodium formation. Mechanistically, lncCCKAR5 served as a scaffold that facilitated the interaction between MKRN2 and LMNA, a key regulator of cytoskeletal function and autophagy. The lncCCKAR5/LMNA/MKRN2 complex played a pivotal role in promoting the ubiquitin-mediated degradation of LMNA, with this effect being further augmented by N6-adenosine methylation of lncCCKAR5. Consequently, our findings underscore the critical role of lncCCKAR5 in regulating the autophagic process in hUCMSCs, particularly through protein ubiquitination and degradation. This intricate regulatory network presents a promising avenue for potential therapeutic interventions in the context of diabetic wound healing involving hUCMSCs.

The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1 dependent axon guidance and branching. Here we demonstrate TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the post-synaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

Fascin structural plasticity mediates flexible actin bundle construction.

Fascin crosslinks actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia and stereocilia. Fascin dysregulation drives aberrant cell migration during metastasis, and fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-electron microscopy, cryo-electron tomography coupled with custom denoising, and computational modeling to probe fascin's F-actin crosslinking mechanisms across spatial scales. Our fascin crossbridge structure reveals an asymmetric F-actin binding conformation that is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements, variability analysis, and simulations show how structural plasticity enables fascin to bridge varied inter-filament orientations, accommodating mismatches between F-actin's helical symmetry and bundle hexagonal packing. Tomography of many-filament bundles and modeling uncovers geometric rules underlying emergent fascin binding patterns, as well as the accumulation of unfavorable crosslinks that limit bundle size. Collectively, this work shows how fascin harnesses fine-tuned nanoscale structural dynamics to build and regulate micron-scale F-actin bundles.

Myo10 tail is crucial for promoting long filopodia.

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

Rosmarinic acid in combination with ginsenoside Rg1 suppresses colon cancer metastasis via co-inhition of COX-2 and PD1/PD-L1 signaling axis.

Metastasis of colorectal cancer (CRC) is a leading cause of mortality among CRC patients. Elevated COX-2 and PD-L1 expression in colon cancer tissue has been linked to distant metastasis of tumor cells. Although COX-2 inhibitors and immune checkpoint inhibitors demonstrate improved anti-tumor efficacy, their toxicity and variable therapeutic effects in individual patients raise concerns. To address this challenge, it is vital to identify traditional Chinese medicine components that modulate COX-2 and PD-1/PD-L1: rosmarinic acid (RA) exerts striking inhibitory effect on COX-2, while ginsenoside Rg1 (GR) possesses the potential to suppress the binding of PD-1/PD-L1. In this study we investigated whether the combination of RA and GR could exert anti-metastatic effects against CRC. MC38 tumor xenograft mouse model with lung metastasis was established. The mice were administered RA (100 mg·kg-1·d-1, i.g.) alone or in combination with GR (100 mg·kg-1·d-1, i.p.). We showed that RA (50, 100, 150 µM) or a COX-2 inhibitor Celecoxib (1, 3, 9 µM) concentration-dependently inhibited the migration and invasion of MC38 cells in vitro. We further demonstrated that RA and Celecoxib inhibited the metastasis of MC38 tumors in vitro and in vivo via interfering with the COX-2-MYO10 signaling axis and inhibiting the generation of filopodia. In the MC38 tumor xenograft mice, RA administration significantly decreased the number of metastatic foci in the lungs detected by Micro CT scanning; RA in combination with GR that had inhibitory effect on the binding of PD-1 and PD-L1 further suppressed the lung metastasis of colon cancer. Compared to COX-2 inhibitors and immune checkpoint inhibitors, RA and GR displayed better safety profiles without disrupting the tissue structures of the liver, stomach and colon, offering insights into the lower toxic effects of clinical traditional Chinese medicine against tumors while retaining its efficacy.

The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.