3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.

Cell invasion is a multi-step process, initiated by the acquisition of a migratory phenotype and the ability to move through complex 3D extracellular environments. We determine the composition of cell-matrix adhesion complexes of invasive breast cancer cells in 3D matrices and identify an interaction complex required for invasive migration. ßPix and myosin18A (Myo18A) drive polarized recruitment of non-muscle myosin 2A (NM2A) to adhesion complexes at the tips of protrusions. Actomyosin force engagement then displaces the Git1-ßPix complex from paxillin, establishing a feedback loop for adhesion maturation. We observe active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. The recruitment of NM2A to adhesions creates a non-muscle myosin isoform gradient, which extends from the protrusion to the nucleus. We postulate that this gradient facilitates coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

Radiolysis-Driven Evolution of Gold Nanostructures - Model Verification by Scale Bridging In Situ Liquid-Phase Transmission Electron Microscopy and X-Ray Diffraction.

Utilizing ionizing radiation for in situ studies in liquid media enables unique insights into nanostructure formation dynamics. As radiolysis interferes with observations, kinetic simulations are employed to understand and exploit beam-liquid interactions. By introducing an intuitive tool to simulate arbitrary kinetic models for radiation chemistry, it is demonstrated that these models provide a holistic understanding of reaction mechanisms. This is shown for irradiated HAuCl4 solutions allowing for quantitative prediction and tailoring of redox processes in liquid-phase transmission electron microscopy (LP-TEM). Moreover, it is demonstrated that kinetic modeling of radiation chemistry is applicable to investigations utilizing X-rays such as X-ray diffraction (XRD). This emphasizes that beam-sample interactions must be considered during XRD in liquid media and shows that reaction kinetics do not provide a threshold dose rate for gold nucleation relevant to LP-TEM and XRD. Furthermore, it is unveiled that oxidative etching of gold nanoparticles depends on both, precursor concentration, and dose rate. This dependency is exploited to probe the electron beam-induced shift in Gibbs free energy landscape by analyzing critical radii of gold nanoparticles.

Laminin N-terminus α31 is upregulated in invasive ductal breast cancer and changes the mode of tumour invasion.

Laminin N-terminus α31 (LaNt α31) is an alternative splice isoform derived from the laminin α3 gene. The LaNt α31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt α31 has never been investigated in a tumour environment. Here we analysed LaNt α31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt α31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt α31 to be upregulated in 56% of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45% of samples. 65.8% of triple negative cases displayed medium to high LaNt α31 expression. To study LaNt α31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. 2D cell migration and invasion into collagen hydrogels were not significantly different between LaNt α31 overexpressing cells and control treated cells. However, LaNt α31 overexpression reduced the proliferation rate of MCF-7 and MDA-MB-231 cells. Moreover, LaNt α31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt α31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt α31 expression could contribute to the processes involved in tumour invasion and may represent a new therapeutic target.

Effect of Bromide on the Surfactant Stabilization Layer Density of Gold Nanorods.

Some studies have speculated that the concentration of bromide ions plays a crucial role in the surfactant density surrounding gold nanorods (AuNR). Small-angle X-ray and neutron scattering (SAXS and SANS) experiments were conducted to analyze any influence the bromide ions might have on the stabilization layer and the aggregation behavior of the ligand CTAB molecules in general. The AuNR were immersed in solutions containing a fixed CTA+ concentration of 2 mM and varying bromide ion concentrations from 0 to 22 mM. A patchy AuNR stabilization shell at low bromide ion concentrations was found, contrary to previously published SANS studies on the AuNR stabilization shell. However, with increasing bromide ion concentration, the density of the stabilization shell increases asymptotically toward a closed/collapsed bilayer configuration. AuNR grown under similar conditions show higher anisotropy with larger bromide ion concentrations. Both results indicate that anisotropic growth strongly depends on a sufficiently dense stabilization layer established by high bromide ion concentrations.

Cells in Slow Motion: Apparent Undercooling Increases Glassy Behavior at Physiological Temperatures.

Solvent conditions are unexpectedly sufficient to drastically and reversibly slow down cells. In vitro on the molecular level, protein-solvent interactions drastically change in the presence of heavy water (D2 O) and its stronger hydrogen bonds. Adding D2 O to the cell medium of living cells increases the molecular intracellular viscosity. While cell morphology and phenotype remain unchanged, cellular dynamics transform into slow motion in a changeable manner. This is exemplified in the slowdown of cell proliferation and migration, which is caused by a reversible gelation of the cytoplasm. In analogy to the time-temperature superposition principle, where temperature is replaced by D2 O, an increase in viscosity slows down the effective time. Actin networks, crucial structures in the cytoplasm, switch from a power-law-like viscoelastic to a more rubber-like elastic behavior. The resulting intracellular resistance and dissipation impair cell movement. Since cells are highly adaptive non-equilibrium systems, they usually respond irreversibly from a thermodynamic perspective. D2 O induced changes, however, are fully reversible and their effects are independent of signaling as well as expression. The stronger hydrogen bonds lead to glass-like, drawn-out intramolecular dynamics, which may facilitate longer storage times of biological matter, for instance, during transport of organ transplants.

Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These N-glycans are predicted to modulate spike binding to the host receptor by stabilizing its open conformation and host immunity evasion. Here, we investigated the essentiality of both the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Ablation of host N-glycosylation using RNA interference or inhibitors, including FDA-approved drugs, reduced the spread of the infection, including that of variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Under these conditions, cells produced fewer virions and some completely lost their infectivity. Furthermore, partial enzymatic deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, we propose protein N-glycosylation as a targetable pathway with clinical potential for treatment of COVID-19. IMPORTANCE The coronavirus SARS-CoV-2 uses its spike surface proteins to infect human cells. Spike proteins are heavily modified with several N-glycans, which are predicted to modulate their function. In this work, we show that interfering with either the synthesis or attachment of spike N-glycans significantly reduces the spread of SARS-CoV-2 infection in vitro, including that of several variants. As new SARS-CoV-2 variants, with various degrees of resistance against current vaccines, are likely to continue appearing, halting virus glycosylation using repurposed human drugs could result in a complementary strategy to reducing the spread of COVID-19 worldwide.

Connecting the dots: combined control of endocytic recycling and degradation.

Endocytosis is an essential process where proteins and lipids are internalised from the plasma membrane in membrane-bound carriers, such as clathrin-coated vesicles. Once internalised into the cell these vesicles fuse with the endocytic network where their contents are sorted towards degradation in the lysosome or recycling to their origin. Initially, it was thought that cargo recycling is a passive process, but in recent years the identification and characterisation of specialised recycling complexes has established a hitherto unthought-of level of complexity that actively opposes degradation. This review will summarise recent developments regarding the composition and regulation of the recycling machineries and their relationship with the degradative pathways of the endosome.

A novel experimental approach for nanostructure analysis: simultaneous small-angle X-ray and neutron scattering.

Exploiting small-angle X-ray and neutron scattering (SAXS/SANS) on the same sample volume at the same time provides complementary nanoscale structural information in two different contrast situations. Unlike an independent experimental approach, the truly combined SAXS/SANS experimental approach ensures the exactness of the probed samples, particularly for in situ studies. Here, an advanced portable SAXS system that is dimensionally suitable for installation in the D22 zone of ILL is introduced. The SAXS apparatus is based on a Rigaku switchable copper/molybdenum microfocus rotating-anode X-ray generator and a DECTRIS detector with a changeable sample-to-detector distance of up to 1.6 m in a vacuum chamber. A case study is presented to demonstrate the uniqueness of the newly established method. Temporal structural rearrangements of both the organic stabilizing agent and organically capped gold colloidal particles during gold nanoparticle growth are simultaneously probed, enabling the immediate acquisition of correlated structural information. The new nano-analytical method will open the way for real-time investigations of a wide range of innovative nanomaterials and will enable comprehensive in situ studies on biological systems. The potential development of a fully automated SAXS/SANS system with a common control environment and additional sample environments, permitting a continual and efficient operation of the system by ILL users, is also introduced.

Membrane Tension Orchestrates Rear Retraction in Matrix-Directed Cell Migration.

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices.

Internal Structure of Nanometer-Sized Droplets Prepared by Antisolvent Precipitation.

Antisolvent precipitation (AP) is a low-cost and less-invasive preparation alternative for organic nanoparticles compared to top-down methods such as high-pressure homogenization or milling. Here we report on particularly small organic nanoparticles (NPs) prepared by AP. It has been found for various materials that these NPs in their liquid state exhibit a significant degree of molecular order at their interface toward the dispersion medium including ubiquinones (coenzyme Q10), triglycerides (trimyristin, tripalmitin), and alkanes (tetracosane). This finding is independent of the use of a stabilizer in the formulation. While this is obviously a quite general interfacial structuring effect, the respective structural details of specific NPs systems might differ. Here, a detailed structural characterization of very small liquid coenzyme Q10 (Q10) NPs is presented as a particular example for this phenomenon. The Q10 NPs have been prepared by AP in the presence of two different stabilizers, sodium dodecyl sulfate (SDS) and pentaethylene glycol monododecyl ether (C12E5), respectively, and without any stabilizer. The NPs' size is initially analyzed by photon correlation spectroscopy (PCS). The SDS-stabilized Q10 NPs have been studied further by differential scanning calorimetry (DSC), small-angle X-ray and neutron scattering (SAXS, SANS), wide-angle X-ray scattering (WAXS), and cryogenic transmission electron microscopy (CryoTEM). A simultaneous analysis of SAXS and contrast variation SANS studies revealed the molecular arrangement within the interface between the NPs and the dispersion medium. The Q10 NPs stabilized by SDS and C12E5, respectively, are small (down to 19.9 nm) and stable (for at least 16 months) even when no stabilizer is used. The SDS-stabilized Q10 NPs reported here, are therewith, to the best of our knowledge, the smallest organic NPs which have been reported to be prepared by AP so far. In particular, these NPs exhibit a core-shell structure consisting of an amorphous Q10 core and a surrounding shell, which is mainly composed of oriented Q10 molecules and aligned SDS molecules. This structure suggests a significant amphiphilic behavior and a rather unexpected stabilizing role of Q10 molecules.

Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis.

Cells going through mitosis undergo precisely timed changes in cell shape and organisation, which serve to ensure the fair partitioning of cellular components into the two daughter cells. These structural changes are driven by changes in actin filament and microtubule dynamics and organisation. While most evidence suggests that the two cytoskeletal systems are remodelled in parallel during mitosis, recent work in interphase cells has implicated the centrosome in both microtubule and actin nucleation, suggesting the potential for regulatory crosstalk between the two systems. Here, by using both in vitro and in vivo assays to study centrosomal actin nucleation as cells pass through mitosis, we show that mitotic exit is accompanied by a burst in cytoplasmic actin filament formation that depends on WASH and the Arp2/3 complex. This leads to the accumulation of actin around centrosomes as cells enter anaphase and to a corresponding reduction in the density of centrosomal microtubules. Taken together, these data suggest that the mitotic regulation of centrosomal WASH and the Arp2/3 complex controls local actin nucleation, which may function to tune the levels of centrosomal microtubules during passage through mitosis.

Actin-Based Cell Protrusion in a 3D Matrix.

Cell migration controls developmental processes (gastrulation and tissue patterning), tissue homeostasis (wound repair and inflammatory responses), and the pathobiology of diseases (cancer metastasis and inflammation). Understanding how cells move in physiologically relevant environments is of major importance, and the molecular machinery behind cell movement has been well studied on 2D substrates, beginning over half a century ago. Studies over the past decade have begun to reveal the mechanisms that control cell motility within 3D microenvironments - some similar to, and some highly divergent from those found in 2D. In this review we focus on migration and invasion of cells powered by actin, including formation of actin-rich protrusions at the leading edge, and the mechanisms that control nuclear movement in cells moving in a 3D matrix.

HRS-WASH axis governs actin-mediated endosomal recycling and cell invasion.

Transmembrane proteins in the sorting endosome are either recycled to their point of origin or destined for lysosomal degradation. Lysosomal sorting is mediated by interaction of ubiquitylated transmembrane proteins with the endosomal sorting complex required for transport (ESCRT) machinery. In this study, we uncover an alternative role for the ESCRT-0 component hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) in promoting the constitutive recycling of transmembrane proteins. We find that endosomal localization of the actin nucleating factor Wiscott-Aldrich syndrome protein and SCAR homologue (WASH) requires HRS, which occupies adjacent endosomal subdomains. Depletion of HRS results in defective constitutive recycling of epidermal growth factor receptor and the matrix metalloproteinase MT1-MMP, leading to their accumulation in internal compartments. We show that direct interactions with endosomal actin are required for efficient recycling and use a model system of chimeric transferrin receptor trafficking to show that an actin-binding motif can counteract an ubiquitin signal for lysosomal sorting. Directed receptor recycling is used by cancer cells to achieve invasive migration. Accordingly, abrogating HRS- and actin-dependent MT1-MMP recycling results in defective matrix degradation and invasion of triple-negative breast cancer cells.

STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap.

The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.

WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion.

Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), which allows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches, we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles in cancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodium assembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturation and degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulates invadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution of Nck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate with high invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP as a hub for signaling molecule recruitment during invadopodium generation and cancer progression, as well as a potential diagnostic biomarker and an optimal target for therapeutic approaches.

PIKfyve, MTMR3 and their product PtdIns5P regulate cancer cell migration and invasion through activation of Rac1.

Previously, we have shown that the phosphoinositide metabolizing enzymes PIKfyve (phosphoinositide 5-kinase, FYVE finger containing) and MTMR3 (myotubularin-related protein 3), together with their lipid product PtdIns5P, are important for migration of normal human fibroblasts. As these proteins are a kinase and a phosphatase respectively, and thereby considered druggable, we wanted to test their involvement in cancer cell migration and invasion. First, we showed that PIKfyve and MTMR3 are expressed in most cancer cells. Next, we demonstrated that depletion of PIKfyve or MTMR3 resulted in decreased velocity in three different cancer cell lines by using new software for cell tracking. Inhibition of the enzymatic activity of PIKfyve by the inhibitor YM201636 also led to a strong reduction in cell velocity. Mechanistically, we show that PIKfyve and MTMR3 regulate the activation of the Rho family GTPase Rac1. Further experiments also implicated PtdIns5P in the activation of Rac1. The results suggest a model for the activation of Rac1 in cell migration where PIKfyve and MTMR3 produce PtdIns5P on cellular membranes which may then serve to recruit effectors to activate Rac1. Finally, in an invasion assay, we demonstrate that both PIKfyve and MTMR3 are implicated in invasive behaviour of cancer cells. Thus PIKfyve and MTMR3 could represent novel therapeutic targets in metastatic cancer.

Cyclical action of the WASH complex: FAM21 and capping protein drive WASH recycling, not initial recruitment.

WASH causes actin to polymerize on vesicles involved in retrograde traffic and exocytosis. It is found within a regulatory complex, but the physiological roles of the other four members are unknown. Here we present genetic analysis of the subunits' individual functions in Dictyostelium. Mutants in each subunit are completely blocked in exocytosis. All subunits except FAM21 are required to drive actin assembly on lysosomes. Without actin, lysosomes never recycle vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) or neutralize to form postlysosomes. However, in FAM21 knockout lysosomes, WASH generates excessive, dynamic streams of actin. These successfully remove V-ATPase, neutralize, and form huge postlysosomes. The distinction between WASH and FAM21 phenotypes is conserved in human cells. Thus, FAM21 and WASH act at different steps of a cyclical pathway in which FAM21 mediates recycling of the complex back to acidic lysosomes. Recycling is driven by FAM21's interaction with capping protein, which couples the WASH complex to dynamic actin on vesicles.

Loss of Scar/WAVE complex promotes N-WASP- and FAK-dependent invasion.

BACKGROUND: The Scar/WAVE regulatory complex (WRC) drives lamellipodia assembly via the Arp2/3 complex, whereas the Arp2/3 activator N-WASP is not essential for 2D migration but is increasingly implicated in 3D invasion. It is becoming ever more apparent that 2D and 3D migration utilize the actin cytoskeletal machinery differently. RESULTS: We discovered that WRC and N-WASP play opposing roles in 3D epithelial cell migration. WRC depletion promoted N-WASP/Arp2/3 complex activation and recruitment to leading invasive edges and increased invasion. WRC disruption also altered focal adhesion dynamics and drove FAK activation at leading invasive edges. We observed coalescence of focal adhesion components together with N-WASP and Arp2/3 complex at leading invasive edges in 3D. Unexpectedly, WRC disruption also promoted FAK-dependent cell transformation and tumor growth in vivo. CONCLUSIONS: N-WASP has a crucial proinvasive role in driving Arp2/3 complex-mediated actin assembly in cooperation with FAK at invasive cell edges, but WRC depletion can promote 3D cell motility.

N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods.

Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation-promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP's arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion.

Actin on trafficking: could actin guide directed receptor transport?

Here, we present emerging ideas surrounding the interplay between the actin cytoskeleton and receptor transport and activation. The bulk of actin dynamics in cells is thought to contribute to architecture and mobility. Actin also contributes to trafficking, acting as a molecular scaffold, providing force to deform membranes, facilitating vesicle abscission or propelling a vesicle through the cytoplasm ( 1) (,) ( 2) and recent studies highlight important connections between the directed trafficking of receptors and the impact on cell migration and actin dynamics. Additionally, a number of newly described actin nucleation promoting factors, such as the vesicle associated protein WASH, reveal unexpected roles of actin in membrane traffic and suggest that the cell dedicates a significant proportion of its regulation of actin dynamics to controlling trafficking.