Cell protrusions and contractions generate long-range membrane tension propagation.

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Filopodia In Vitro and In Vivo.

Filopodia are dynamic cell surface protrusions used for cell motility, pathogen infection, and tissue development. The molecular mechanisms determining how and where filopodia grow and retract need to integrate mechanical forces and membrane curvature with extracellular signaling and the broader state of the cytoskeleton. The involved actin regulatory machinery nucleates, elongates, and bundles actin filaments separately from the underlying actin cortex. The refined membrane and actin geometry of filopodia, importance of tissue context, high spatiotemporal resolution required, and high degree of redundancy all limit current models. New technologies are improving opportunities for functional insight, with reconstitution of filopodia in vitro from purified components, endogenous genetic modification, inducible perturbation systems, and the study of filopodia in multicellular environments. In this review, we explore recent advances in conceptual models of how filopodia form, the molecules involved in this process, and our latest understanding of filopodia in vitro and in vivo.

Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Allosteric transcription stimulation by RNA polymerase II super elongation complex.

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila.

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell-cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.

Primordial germ cells adjust their protrusion type while migrating in different tissue contexts in vivo.

In both physiological processes and disease contexts, migrating cells have the ability to adapt to conditions in their environment. As an in vivo model for this process, we use zebrafish primordial germ cells that migrate throughout the developing embryo. When migrating within an ectodermal environment, the germ cells form fewer and smaller blebs when compared with their behavior within mesodermal environment. We find that cortical tension of neighboring cells is a parameter that affects blebbing frequency. Interestingly, the change in blebbing activity is accompanied by the formation of more actin-rich protrusions. These alterations in cell behavior that correlate with changes in RhoA activity could allow the cells to maintain dynamic motility parameters, such as migration speed and track straightness, in different settings. In addition, we find that the polarity of the cells can be affected by stiff structures positioned in their migration path This article has an associated 'The people behind the papers' interview.

Geometry-mediated bridging drives nonadhesive stripe wound healing.

Wound healing through reepithelialization of gaps is of profound importance to the medical community. One critical mechanism identified by researchers for closing non-cell-adhesive gaps is the accumulation of actin cables around concave edges and the resulting purse-string constriction. However, the studies to date have not separated the gap-edge curvature effect from the gap size effect. Here, we fabricate micropatterned hydrogel substrates with long, straight, and wavy non-cell-adhesive stripes of different gap widths to investigate the stripe edge curvature and stripe width effects on the reepithelialization of Madin-Darby canine kidney (MDCK) cells. Our results show that MDCK cell reepithelization is closely regulated by the gap geometry and may occur through different pathways. In addition to purse-string contraction, we identify gap bridging either via cell protrusion or by lamellipodium extension as critical cellular and molecular mechanisms for wavy gap closure. Cell migration in the direction perpendicular to wound front, sufficiently small gap size to allow bridging, and sufficiently high negative curvature at cell bridges for actin cable constriction are necessary/sufficient conditions for gap closure. Our experiments demonstrate that straight stripes rarely induce cell migration perpendicular to wound front, but wavy stripes do; cell protrusion and lamellipodia extension can help establish bridges over gaps of about five times the cell size, but not significantly beyond. Such discoveries deepen our understanding of mechanobiology of cell responses to curvature and help guide development of biophysical strategies for tissue repair, plastic surgery, and better wound management.

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.

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.

Tunneling nanotubes and tumor microtubes-Emerging data on their roles in intercellular communication and pathophysiology: Summary of an International FASEB Catalyst Conference October 2023.

In the past decade, there has been a steady rise in interest in studying novel cellular extensions and their potential roles in facilitating human diseases, including neurologic diseases, viral infectious diseases, cancer, and others. One of the exciting new aspects of this field is improved characterization and understanding of the functions and potential mechanisms of tunneling nanotubes (TNTs), which are actin-based filamentous protrusions that are structurally distinct from filopodia. TNTs form and connect cells at long distance and serve as direct conduits for intercellular communication in a wide range of cell types in vitro and in vivo. More researchers are entering this field and investigating the role of TNTs in mediating cancer cell invasion and drug resistance, cellular transfer of proteins, RNA or organelles, and intercellular spread of infectious agents, such as viruses, bacteria, and prions. Even further, the elucidation of highly functional membrane tubes called "tumor microtubes" (TMs) in incurable gliomas has further paved a new path for understanding how and why the tumor type is highly invasive at the cellular level and also resistant to standard therapies. Due to the wide-ranging and rapidly growing applicability of TNTs and TMs in pathophysiology across the spectrum of biology, it has become vital to bring researchers in the field together to discuss advances and the future of research in this important niche of protrusion biology.

Involvement of cellular protrusions in gamete interactions.

Gamete fusion is of considerable importance in reproductive events, as it determines the gamete pairs or chromosomes that the next generation will inherit. To preserve species specificity with an appropriate karyotype, the fusion between gametes requires regulatory mechanisms to ensure limited fusion competency. In many organisms, gamete surfaces are not smooth, but present constitutive or transient cellular protrusions suggested to be involved in gamete fusion. However, the molecular mechanisms and the factors essential for the membrane-membrane fusion process and cellular protrusion involvement have remained unclear. Recent advances in the identification and functional analysis of the essential factors for gamete interaction have revealed the molecular mechanisms underlying their activity regulation and dynamics. In homogametic fertilization, dynamic regulation of the fusion core machinery on cellular protrusions was precisely uncovered. In heterogametic fertilization, oocyte fusion competency was suggested to correlate with the compartmentalization of the fusion essential factor and protrusion formation. These findings shed light on the significance of cellular protrusions in gamete fusion as a physically and functionally specialized site for cellular fusion. In this review, we consider the developments in gamete interaction research in various species with different fertilization modes, highlighting the commonalities in the relationship between gamete fusion and cellular protrusions.

Removal of cellular protrusions.

Cell-cell communications are central to a variety of physiological and pathological processes in multicellular organisms. Cells often rely on cellular protrusions to communicate with one another, which enable highly selective and efficient signaling within complex tissues. Owing to significant improvements in imaging techniques, identification of signaling protrusions has increased in recent years. These protrusions are structurally specialized for signaling and facilitate interactions between cells. Therefore, physical regulation of these structures must be key for the appropriate strength and pattern of signaling outcomes. However, the typical approaches for understanding signaling regulation tend to focus solely on changes in signaling molecules, such as gene expression, protein-protein interaction, and degradation. In this short review, we summarize the studies proposing the removal of different types of signaling protrusions-including cilia, neurites, MT (microtubule based)-nanotubes and microvilli-and discuss their mechanisms and significance in signaling regulation.

WIV, a protein domain found in a wide number of arthropod viruses, which probably facilitates infection.

The most powerful approach to detect distant homologues of a protein is based on structure prediction and comparison. Yet this approach is still inapplicable to many viral proteins. Therefore, we applied a powerful sequence-based procedure to identify distant homologues of viral proteins. It relies on three principles: (1) traces of sequence similarity can persist beyond the significance cutoff of homology detection programmes; (2) candidate homologues can be identified among proteins with weak sequence similarity to the query by using 'contextual' information, e.g. taxonomy or type of host infected; (3) these candidate homologues can be validated using highly sensitive profile-profile comparison. As a test case, this approach was applied to a protein without known homologues, encoded by ORF4 of Lake Sinai viruses (which infect bees). We discovered that the ORF4 protein contains a domain that has homologues in proteins from >20 taxa of viruses infecting arthropods. We called this domain 'widespread, intriguing, versatile' (WIV), because it is found in proteins with a wide variety of functions and within varied domain contexts. For example, WIV is found in the NSs protein of tospoviruses, a global threat to food security, which infect plants as well as their arthropod vectors; in the RNA2 ORF1-encoded protein of chronic bee paralysis virus, a widespread virus of bees; and in various proteins of cypoviruses, which infect the silkworm Bombyx mori. Structural modelling with AlphaFold indicated that the WIV domain has a previously unknown fold, and bibliographical evidence suggests that it facilitates infection of arthropods.

Analysis of monocyte cell tractions in 2.5D reveals mesoscale mechanics of podosomes during substrate-indenting cell protrusion.

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.

Ena/VASP proteins in cell edge protrusion, migration and adhesion.

The tightly coordinated, spatiotemporal control of actin filament remodeling provides the basis of fundamental cellular processes, such as cell migration and adhesion. Specific protein assemblies, composed of various actin-binding proteins, are thought to operate in these processes to nucleate and elongate new filaments, arrange them into complex three-dimensional (3D) arrays and recycle them to replenish the actin monomer pool. Actin filament assembly is not only necessary to generate pushing forces against the leading edge membrane or to propel pathogens through the cytoplasm, but also coincides with the generation of stress fibers (SFs) and focal adhesions (FAs) that generate, transmit and sense mechanical tension. The only protein families known to date that directly enhance the elongation of actin filaments are formins and the family of Ena/VASP proteins. Their mechanisms of action, however, in enhancing processive filament elongation are distinct. The aim of this Review is to summarize our current knowledge on the molecular mechanisms of Ena/VASP-mediated actin filament assembly, and to discuss recent insights into the cell biological functions of Ena/VASP proteins in cell edge protrusion, migration and adhesion.

Lamellipodia-like actin networks in cells lacking WAVE regulatory complex.

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.

Deploy the proboscis!: Functional morphology and kinematics of a novel form of extreme jaw protrusion in the hingemouth, Phractolaemus ansorgii (Gonorynchiformes).

Premaxillary protrusion and the performance advantages it confers are implicated in the success of diverse lineages of teleost fishes, such as Cypriniformes and Acanthomorpha. Although premaxillary protrusion has evolved independently at least five times within bony fishes, much of the functional work investigating this kinesis relates to mechanisms found only in these two clades. Few studies have characterized feeding mechanisms in less-diverse premaxilla-protruding lineages and fewer yet have investigated the distinctive anatomy underlying jaw kinesis in these lineages. Here, we integrated dissection, clearing and staining, histology, micro-CT, and high-speed videography to investigate an isolated and independent origin of jaw protrusion in the hingemouth, Phractolaemus ansorgii, which employs a complex arrangement of bones, musculature, and connective tissues to feed on benthic detritus via a deployable proboscis. Our goals were to provide an integrative account of the underlying architecture of P. ansorgii's feeding apparatus and to assess the functional consequences of this drastic deviation from the more typical teleost condition. Phractolaemus ansorgii's cranial anatomy is distinct from all other fishes in that its adducted lower jaw is caudally oriented, and it possesses a mouth at the terminal end of an elongated, tube-like proboscis that is unique in its lack of skeletal support from the oral jaws. Instead, its mouth is supported primarily by hyaline-cell cartilage and other rigid connective tissues, and features highly flexible lips that are covered in rows of keratinous unculi. Concomitant changes to the adductor musculature likely allow for the flexibility to protrude the mouth dorsally and ventrally as observed during different feeding behaviors, while the intrinsic compliance of the lips allows for more effective scraping of irregular surfaces. From our feeding videos, we find that P. ansorgii is capable of modulating the distance of protrusion, with maximum anterior protrusion exceeding 30% of head length. This represents a previously undescribed example of extreme jaw protrusion on par with many acanthomorph species. Protrusion is much slower in P. ansorgii-reaching an average speed of 2.74 cm/s-compared to acanthomorphs feeding on elusive prey or even benthivorous cypriniforms. However, this reorganization of cranial anatomy may reflect a greater need for dexterity to forage more precisely in multiple directions and on a wide variety of surface textures. Although this highly modified mechanism may have limited versatility over evolutionary timescales, it has persisted in solitude within Gonorynchiformes, representing a novel functional solution for benthic feeding in tropical West African rivers.

Silver nanowires/waterborne polyurethane composite film based piezoresistive pressure sensor for ultrasensitive human motion monitoring.

Flexible piezoresistive pressure sensors are gaining significant attention, particularly in the realm of flexible wearable electronic skin. Here, a flexible piezoresistive pressure sensor was developed with a broad sensing range and high sensitivity. We achieved this by curing polydimethylsiloxane (PDMS) on sandpaper, creating a PDMS film as the template with a micro-protrusion structure. The core sensing layer was formed using a composite of silver nanowires (AgNWs) and waterborne polyurethane (WPU) with a similar micro-protrusion structure. The sensor stands out with its exceptional sensitivity, showing a value of 1.04 × 106kPa-1with a wide linear range from 0 to 27 kPa. It also boasts a swift response and recovery time of 160 ms, coupled with a low detection threshold of 17 Pa. Even after undergoing more than 1000 cycles, the sensor continues to deliver stable performance. The flexible piezoresistive pressure sensor based on AgNWs/WPU composite film (AWCF) can detect small pressure changes such as pulse, swallowing, etc, which indicates that the sensor has great application potential in monitoring human movement and flexible wearable electronic skin.

Novel clinical diagnostic criteria based on noninvasive examination findings to predict the presence of urodynamic detrusor underactivity in men.

AIMS: To investigate the usefulness of novel clinical diagnostic criteria based on noninvasive examination findings to diagnose urodynamic detrusor underactivity (DU) in men. METHODS: We developed clinical diagnostic criteria to predict the presence of urodynamic DU in men as follows: (a) bladder voiding efficiency <70% on uroflowmetry, (b) existence of "sawtooth and interrupted waveforms" on uroflowmetry, and (c) ultrasonography-documented intravesical prostatic protrusion <10 mm. We analyzed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of these clinical criteria for diagnosing urodynamic DU in men aged 50 years or above with lower urinary tract symptoms who underwent urodynamic studies. RESULTS: Of the 314 men analyzed (mean age, 72.4 years; mean detrusor contraction index [DCI], 98.8; and mean bladder outlet obstruction index [BOOI], 43.9), 89 men met this clinical DU diagnostic criteria. Of these, 79 men (88.8%) had urodynamic DU (DCI < 100 and BOOI < 40), nine (10.1%) had DU + BOO (DCI < 100 and BOOI ≥ 40), and one (1.1%) had normal voiding functions. None of the men with urodynamic BOO (DCI ≥ 100 and BOOI ≥ 40) met the clinical DU diagnostic criteria. The sensitivity, specificity, PPV, and NPV of these clinical diagnostic criteria for urodynamic DU were 69.3%, 95.0%, 88.8%, and 84.4%, respectively. CONCLUSION: The proposed clinical DU diagnostic criteria showed a high PPV (88.8%) for diagnosing urodynamic DU. None of the patients with BOO met the clinical diagnostic criteria for DU. These clinical DU diagnostic criteria may be useful in identifying men with urodynamic DU in clinical practice.

Molecular Pathways and Animal Models of Atrioventricular Septal Defect.

The development of a fully functional four-chambered heart is critically dependent on the correct formation of the structures that separate the atrial and ventricular chambers. Perturbation of this process typically results in defects that allow mixing of oxygenated and deoxygenated blood. Atrioventricular septal defects (AVSD) form a class of congenital heart malformations that are characterized by the presence of a primary atrial septal defect (pASD), a common atrioventricular valve (cAVV), and frequently also a ventricular septal defect (VSD). While AVSD were historically considered to result from failure of the endocardial atrioventricular cushions to properly develop and fuse, more recent studies have determined that inhibition of the development of other components of the atrioventricular mesenchymal complex can lead to AVSDs as well. The role of the dorsal mesenchymal protrusion (DMP) in AVSD pathogenesis has been well-documented in studies using animal models for AVSDs, and in addition, preliminary data suggest that the mesenchymal cap situated on the leading edge of the primary atrial septum may be involved in certain situations as well. In this chapter, we review what is currently known about the molecular mechanisms and animal models that are associated with the pathogenesis of AVSD.