Directional Motility Research Articles

Patch-Based Far-Infrared Radiation (FIR) Therapy Does Not Impact Cell Tracking or Motility of Human Melanoma Cells In Vitro.

Far-Infrared Radiation (FIR) is emerging as a novel non-invasive tool for mitigating inflammation and oxidative stress, offering potential benefits for certain medical conditions such as cardiovascular disease and chronic inflammatory disorders. We previously demonstrated that the application of patch-based FIR therapy on human umbilical vein endothelial cells (HUVECs) reduced the expression of inflammatory biomarkers and the levels of reactive oxygen species (ROS). Several in vitro studies have shown the inhibitory effects of FIR therapy on cell growth in different cancer cells (including murine melanoma cells), mainly using the wound healing assay, without direct cell motility or tracking analysis. The main objective of the present study was to conduct an in-depth analysis of single-cell motility and tracking during the wound healing assay, using an innovative high-throughput technique in the human melanoma cell line M14/C2. This technique evaluates various motility descriptors, such as average velocity, average curvature, average turning angle, and diffusion coefficient. Our results demonstrated that patch-based FIR therapy did not impact cell proliferation and viability or the activation of mitogen-activated protein kinases (MAPKs) in the human melanoma cell line M14/C2. Moreover, no significant differences in cell motility and tracking were observed between control cells and patch-treated cells. Altogether, these findings confirm the beneficial effects of the in vitro application of patch-based FIR therapy in human melanoma cell lines, although such effects need to be confirmed in future in vivo studies.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart directional motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced enhanced motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

The role of TGF-β in the electrotactic reaction of mouse 3T3 fibroblasts in vitro.

Endogenous electric fields (EFs) serve as a crucial signal to guide cell movement in processes such as wound healing, embryonic development, and cancer metastasis. However, the mechanism underlying cell electrotaxis remains poorly understood. A plausible hypothesis suggests that electrophoretic or electroosmotic forces may rearrange charged components of the cell membrane, including receptors for chemoattractants which induce asymmetric signaling and directional motility. This study aimed to explore the role of Transforming Growth Factor Beta (TGFβ) signaling in the electrotactic reaction of 3T3 fibroblasts. Our findings indicate that inhibiting canonical and several non-canonical signaling pathways originating from the activated TGF-β receptor does not hinder the directed migration of 3T3 cells to the cathode. Furthermore, suppression of TGF-β receptor expression does not eliminate the directional migration effect of 3T3 cells in the electric field. Additionally, there is no observed redistribution of the TGF-β receptor in the electric field. However, our studies affirm the significant involvement of Phosphoinositide 3-Kinase (PI3K) in electrotaxis, suggesting that in our model, its activation is likely associated with factors independent of TGFβ action.

In situ enzymatic control of colloidal phoresis and catalysis through hydrolysis of ATP.

The ability to sense chemical gradients and respond with directional motility and chemical activity is a defining feature of complex living systems. There is a strong interest among scientists to design synthetic systems that emulate these properties. Here, we realize and control such behaviors in a synthetic system by tailoring multivalent interactions of adenosine nucleotides with catalytic microbeads. We first show that multivalent interactions of the bead with gradients of adenosine mono-, di- and trinucleotides (AM/D/TP) control both the phoretic motion and a proton-transfer catalytic reaction, and find that both effects are diminished greatly with increasing valence of phosphates. We exploit this behavior by using enzymatic hydrolysis of ATP to AMP, which downregulates multivalent interactivity in situ. This produces a sudden increase in transport of the catalytic microbeads (a phoretic jump), which is accompanied by increased catalytic activity. Finally, we show how this enzymatic activity can be systematically tuned, leading to simultaneous in situ spatial and temporal control of the location of the microbeads, as well as the products of the reaction that they catalyze. These findings open up new avenues for utilizing multivalent interaction-mediated programming of complex chemo-mechanical behaviors into active systems.

Systemic cellular migration: The forces driving the directed locomotion movement of cells.

Directional motility is an essential property of cells. Despite its enormous relevance in many fundamental physiological and pathological processes, how cells control their locomotion movements remains an unresolved question. Here, we have addressed the systemic processes driving the directed locomotion of cells. Specifically, we have performed an exhaustive study analyzing the trajectories of 700 individual cells belonging to three different species (Amoeba proteus, Metamoeba leningradensis, and Amoeba borokensis) in four different scenarios: in absence of stimuli, under an electric field (galvanotaxis), in a chemotactic gradient (chemotaxis), and under simultaneous galvanotactic and chemotactic stimuli. All movements were analyzed using advanced quantitative tools. The results show that the trajectories are mainly characterized by coherent integrative responses that operate at the global cellular scale. These systemic migratory movements depend on the cooperative nonlinear interaction of most, if not all, molecular components of cells.

Hemocytes from neonates of Biomphalaria glabrata are functionally less mature than those from adult snails

AbstractGastropod molluscs, which serve as obligatory intermediate hosts for digenetic trematodes, possess an internal defense system (IDS) consisting of phagocytic hemocytes and plasma factors. This IDS is responsible for resistance to infection with larval trematodes, which are encapsulated and killed by hemocytes in incompatible snails. Like other physiological systems, the IDS probably undergoes maturation during early stages of life, and the relatively undeveloped state of the IDS in young snails has been hypothesized to be a factor in their increased susceptibility to infection with larval trematodes. In this study, hemocytes were examined in the BS‐90 laboratory strain of Biomphalaria glabrata that is resistant to infection with Schistosoma mansoni as adults but susceptible to infection as neonates. Compared with hemocytes from adults, hemocytes from neonates had a smaller perimeter and lower intrinsic directional motility on glass microscope slides. Additionally, in vitro assays showed a lower association with fucoidan‐linked polystyrene beads and less ability to produce superoxide anion in hemocytes from neonates compared to hemocytes from adults. These results support the hypothesis that the gastropod IDS undergoes maturation during growth. However, whether the observed differences between hemocytes of neonatal and adult BS‐90 snails play a role in the susceptibility of the former and resistance of the latter to infection with S. mansoni is not known.

A Study on the Bacteriological Profile of Ascitic Fluids and Their Antibiotic Susceptibility Pattern in a Tertiary Care Hospital.

Background Ascitic fluid culture remains an essential step in the management of all patients with ascites, regardless of their presenting complaints. Diagnostic paracentesis should not be delayed or prevent timely administration of antibiotics, particularly in unstable patients. Hence, it is an essential part of the surveillance system of every hospital to perform ascitic fluid culture and assess the antibiotic susceptibility patterns of bacterial isolates. In view of this perspective, the present study was conducted at Chengalpattu Medical College Hospital, Tamil Nadu, India. Objective The aim of the study isto determine the bacterial isolatesof ascitic fluid samples and study their antibiotic susceptibility patterns. Materials and methods Ascitic fluids received in the central laboratory at the Department of Microbiology from various departments were included in this study. Preliminary identification of isolates wasperformed by direct Gram staining, acid-fast staining, and motility testing by the hanging drop method. Within one hour of receiving the samples, they were plated onto blood agar and MacConkey agar media and incubated for 18-24 hours at 37°C for isolation. Growth was checked, and species identification was done based on conventional methods. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Results In this study, a total of 100 ascitic fluid samples were collected, of which only eight (8%) showed growth. Among the eight isolates, six (75%) were Gram-negative bacilli (GNB). Four (66.66%) of the six GNB wereKlebsiella spp., while the remaining two (33.33%) were Escherichia coli. Both Gram-positive cocci wereStaphylococcus aureus. All the GNB isolates were susceptible to meropenem, piperacillin-tazobactam, and ceftriaxone, with varying susceptibilities to other drugs. Both Gram-positive isolates were found to be methicillin-sensitive Staphylococcus aureus. Conclusion GNB were the predominant organisms in cases of ascitic fluid infection, and they showed 100% susceptibility to carbapenem drugs (especially meropenem),piperacillin-tazobactam, and ceftriaxone.All these drugs can be kept in reserve for serious infections. Amikacin and gentamicin showed promising susceptibility. These drugs can be started empirically with patients on admission before performing culture. Drug adjustments may be later made based on culture reports.

Motility of Synthetic Cells from Engineered Lipids.

Synthetic cells are artificial systems that resemble natural cells. Significant efforts have been made over the years to construct synthetic protocells that can mimic biological mechanisms and perform various complex processes. These include compartmentalization, metabolism, energy supply, communication, and gene reproduction. Cell motility is also of great importance, as nature uses elegant mechanisms for intracellular trafficking, immune response, and embryogenesis. In this review, we discuss the motility of synthetic cells made from lipid vesicles and relevant molecular mechanisms. Synthetic cell motion may be classified into surface-based or solution-based depending on whether it involves interactions with surfaces or movement in fluids. Collective migration behaviors have also been demonstrated. The swarm motion requires additional mechanisms for intercellular signaling and directional motility that enable communication and coordination among the synthetic vesicles. In addition, intracellular trafficking for molecular transport has been reconstituted in minimal cells with the help of DNA nanotechnology. These efforts demonstrate synthetic cells that can move, detect, respond, and interact. We envision that new developments in protocell motility will enhance our understanding of biological processes and be instrumental in bioengineering and therapeutic applications.

Characterization of a sperm motility signalling pathway in a gonochoric coral suggests conservation across cnidarian sexual systems.

Most stony corals liberate their gametes into the water column via broadcast spawning, where fertilization hinges upon the activation of directional sperm motility. Sperm from gonochoric and hermaphroditic corals display distinct morphological and molecular phenotypes, yet it is unknown whether the signalling pathways controlling sperm motility are also distinct between these sexual systems. Here, we addressed this knowledge gap using the gonochoric, broadcast spawning coral Astrangia poculata. We found that cytosolic alkalinization of sperm activates the pH-sensing enzyme soluble adenylyl cyclase (sAC), which is required for motility. Additionally, we demonstrate for the first time in any cnidarian that sAC activity leads to protein kinase A (PKA) activation, and that PKA activity contributes to sperm motility activation. Ultrastructures of A. poculata sperm displayed morphological homology with other gonochoric cnidarians, and sAC exhibited broad structural and functional conservation across this phylum. These results indicate a conserved role for pH-dependent sAC-cAMP-PKA signalling in sperm motility across coral sexual systems, and suggest that the role of this pathway in sperm motility may be ancestral in metazoans. Finally, the dynamics of this pH-sensitive pathway may play a critical role in determining the sensitivity of marine invertebrate reproduction to anthropogenic ocean acidification.

Editorial for the special issue “Control‐theoretic approaches for systems in the life sciences”

Editorial for the special issue “Control<scp>‐theoretic</scp> approaches for systems in the life sciences”

The Role of Adhesion in Helicobacter pylori Persistent Colonization.

Helicobacter pylori (H. pylori) has coevolved with its human host for more than 100 000years. It can safely colonize around the epithelium of gastric glands via their specific microstructures and proteins. Unless patients receive eradication treatment, H. pylori infection is always lifelong. However, few studies have discussed the reasons. This review will focus on the adhesion of H. pylori from the oral cavity to gastric mucosa and summarize the possible binding and translocation characteristics. Adhesion is the first step for persistent colonization after the directional motility, and factors related to adhesion are necessary. Outer membrane proteins, such as the blood group antigen binding adhesin (BabA) and the sialic acid binding adhesin (SabA), play pivotal roles in binding to human mucins and cellular surfaces. And this may offer different perspectives on eradication.

Differential contractility regulates cancer stem cell migration

Cancer stem cells (CSCs) are known to have a high capacity for tumor initiation and the formation of metastases. We have previously shown that in collagen constructs mimetic of aligned extracellular matrix architectures observed in carcinomas, breast CSCs demonstrate enhanced directional and total motility compared with more differentiated carcinoma populations. Here, we show that CSCs maintain increased motility in diverse environments including on 2D elastic polyacrylamide gels of various stiffness, 3D randomly oriented collagen matrices, and ectopic cerebral slices representative of a common metastatic site. A consistent twofold increase of CSC motility across platforms suggests a general shift in cell migration mechanics between well-differentiated carcinoma cells and their stem-like counterparts. To further elucidate the source of differences in migration, we demonstrate that CSCs are less contractile than the whole population (WP) and develop fewer and smaller focal adhesions and show that enhanced CSC migration can be tuned via contractile forces. The WP can be shifted to a CSC-like migratory phenotype using partial myosin II inhibition. Inversely, CSCs can be shifted to a less migratory WP-like phenotype using microtubule-destabilizing drugs that increase contractility or by directly enhancing contractile forces. This work begins to reveal the mechanistic differences driving CSC migration and raises important implications regarding the potentially disparate effects of microtubule-targeting agents on the motility of different cell populations.

Abstract A017: Polyaneuploid Cancer Cells (PACCs) as metastasis-competent cells

Abstract Metastasis is responsible for 90% of cancer deaths, yet on a cellular level, successful metastasis is rare. Barriers to metastasis exist at every step of the metastatic cascade (invasion, intravasation, survival in the circulation, extravasation, and colonization.) Modeling predicts that only one of every 1.5 billion circulating tumor cells results in clinically-detectable metastasis. Identification of the rare subset of metastasis-competent cells is the most crucial goal of cancer research. Polyanuploid Cancer Cells (PACCs) are physically enlarged, treatment-resistant cells with increased genomic content that form in response to stress. After stress-induction, PACCs exist in a non-proliferative state for many weeks before undergoing eventual depolyploidization to produce progeny of “typical” cancer cell size, morphology, and nuclear content. PACCs can be identified in both primary and metastatic patient tumor tissues. As such, we propose Polyaneuploid Cancer Cells (PACCs) are metastasis-competent cells. Single-cell tracking reveals that PACCs are more motile than nonPACCs. Additionally, we observe that PACCs exhibit increased directional migration in 2D chemotactic environments. Optical-tracking of spontaneous bead motion reveals that PACCs demonstrate increased cytoskeletal rearrangement, an observation that aligns with increased environment-sensing and directional motility. In total, this predicts successful invasion. Analyses of cellular deformability using Magnetic Twisting Cytometry and Atomic Force Microscopy jointly reveal that cells in the PACC state display hyper-elastic properties. Among these include increased peripheral deformability and maintained peri-nuclear cortical integrity, both of which predict successful intravasation and extravasation. Functional deformability of PACCs navigating through narrow channels in a chemotactic environment was confirmed using a custom microfluids device. RTqPCR, NanoString mRNA quantification, Western blot, and immunofluorescent imaging reveal that PACCs highly overexpress Vimentin, a cytoskeletal component known to confer hyper-elasticity. Notably, there is no correlation between Vimentin content and motility dynamics in PACCs, indicating that the role of Vimentin in PACCs may primarily drive increased hyper-elasticity rather than increased motility. Anoikis-resistance assays and detection of PACCs in the blood of a patient with metastatic prostate cancer using a selection-free circulating tumor cell detection platform reveal that PACCs are capable of surviving in the circulation. Our work to date reports that PACCs demonstrate increased motility, environment-sensing, hyper-elasticity, and anoikis-resistance. Taken together with the knowledge that PACCs exist in a treatment-resistant state and are capable of eventual depolyploidization (as a potential route to successful colony formation), this data suggests PACCs are a candidate rare metastasis-competent cell type worthy of further analysis. Citation Format: Mikaela M. Mallin, Nicholas Kim, Mohammad Ikbal Choudhury, SeJong Lee, Steven S. An, Sean X. Sun, Konstantinos Konstantopoulos, Sarah R. Amend, Kenneth J. Pienta. Polyaneuploid Cancer Cells (PACCs) as metastasis-competent cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A017.

The Vibrio Polar Flagellum: Structure and Regulation.

Here we discuss the structure and regulation of the Vibrio flagellum and its role in the virulence of pathogenic species. We will cover some of the novel insights into the structure of this nanomachine that have recently been enabled by cryoelectron tomography. We will also highlight the recent genetic studies that have increased our understanding in flagellar synthesis specifically at the bacterial cell pole, temporal regulation of flagellar genes, and how the flagellum enables directional motility through Run-Reverse-Flick cycles.

Impact of direct physical association and motility on fitness of a synthetic interkingdom microbial community

Mutualistic exchange of metabolites can play an important role in microbial communities. Under natural environmental conditions, such exchange may be compromised by the dispersal of metabolites and by the presence of non-cooperating microorganisms. Spatial proximity between members during sessile growth on solid surfaces has been shown to promote stabilization of cross-feeding communities against these challenges. Nonetheless, many natural cross-feeding communities are not sessile but rather pelagic and exist in turbulent aquatic environments, where partner proximity is often achieved via direct cell-cell adhesion, and cooperation occurs between physically associated cells. Partner association in aquatic environments could be further enhanced by motility of individual planktonic microorganisms. In this work, we establish a model bipartite cross-feeding community between bacteria and yeast auxotrophs to investigate the impact of direct adhesion between prokaryotic and eukaryotic partners and of bacterial motility in a stirred mutualistic co-culture. We demonstrate that adhesion can provide fitness benefit to the bacterial partner, likely by enabling local metabolite exchange within co-aggregates, and that it counteracts invasion of the community by a non-cooperating cheater strain. In a turbulent environment and at low cell densities, fitness of the bacterial partner and its competitiveness against a non-cooperating strain are further increased by motility that likely facilitates partner encounters and adhesion. These results suggest that, despite their potential fitness costs, direct adhesion between partners and its enhancement by motility may play key roles as stabilization factors for metabolic communities in turbulent aquatic environments.

The βI domain promotes active β1 integrin clustering into mature adhesion sites.

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1-containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain-dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone.

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei.

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum's tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2's effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids' unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally.

Roles of the nucleus in leukocyte migration.

Leukocytes patrol our bodies in search of pathogens and migrate to sites of injury in response to various stimuli. Rapid and directed leukocyte motility is therefore crucial to our immunity. The nucleus is the largest and stiffest cellular organelle and a mechanical obstacle for migration through constrictions. However, the nucleus is also essential for 3D cell migration. Here, we review the roles of the nucleus in leukocyte migration, focusing on how cells deform their nuclei to aid cell motility and the contributions of the nucleus to cell migration. We discuss the regulation of the nuclear biomechanics by the nuclear lamina and how it, together with the cytoskeleton, modulates the shapes of leukocyte nuclei. We then summarize the functions of nesprins and SUN proteins in leukocytes and discuss how forces are exerted on the nucleus. Finally, we examine the mechanical roles of the nucleus in cell migration, including its roles in regulating the direction of migration and path selection.

Fibronectin anchoring to viscoelastic poly(dimethylsiloxane) elastomers controls fibroblast mechanosensing and directional motility

The established link between deregulated tissue mechanics and various pathological states calls for the elucidation of the processes through which cells interrogate and interpret the mechanical properties of their microenvironment. In this work, we demonstrate that changes in the presentation of the extracellular matrix protein fibronectin on the surface of viscoelastic silicone elastomers have an overarching effect on cell mechanosensing, that is independent of bulk mechanics. Reduction of surface hydrophilicity resulted in altered fibronectin adsorption strength as monitored using atomic force microscopy imaging and pulling experiments. Consequently, primary human fibroblasts were able to remodel the fibronectin coating, adopt a polarized phenotype and migrate directionally even on soft elastomers, that otherwise were not able to resist the applied traction forces. The findings presented here provide valuable insight on how cellular forces are regulated by ligand presentation and used by cells to probe their mechanical environment, and have implications on biomaterial design for cell guidance.

Mechanical Tension‐induced Vascular and Alveolar Morphogenesis during Lung Regeneration

Mechanical forces altered after unilateral pneumonectomy (PNX) control post‐PNX vascular and alveolar morphogenesis. This study aims to understand the mechanosensitive mechanism of lung vascular and alveolar regeneration in the post‐PNX mouse lungs. Vascular formation is stimulated at the subpleural region in the remaining mouse lung after unilateral PNX, where mechanical stretch is enhanced, while inhibition of mechanical tension by insertion of prosthesis attenuates these effects. The focal adhesion protein, paxillin senses mechanical stretch after PNX and the levels of paxillin are upregulated in SPB‐positive alveolar epithelial type II (ATII) cells and endothelial cells (ECs), which stimulates the expression of angiogenic factor angiopoietin‐1 (Ang1) in ATII cells and Ang2 in ECs to induce EC directional motility to the subpleural region. Cellular stretching increases the expression of paxillin, which induces expression of Ang2 in ECs and Ang1 in ATII cells in vitro. Post‐PNX lung growth and vascular formation toward the subpleural region are suppressed in Pxnfl/fl‐Sftpc‐CreERT2 mouse lungs, in which paxillin expression is attenuated in ATII cells. Structure of lamellar bodies that control surfactant homeostasis is also disrupted in post‐PNX Pxnfl/fl‐Sftpc‐CreERT2 mouse lung ATII cells. These results suggest that cooperative action of mechanosensitive paxillin signaling in ECs and ATII cells mediates vascular and alveolar epithelial formation during post‐PNX lung growth.