Motile Processes Research Articles

Mitochondria are absent from microglial processes performing surveillance, chemotaxis, and phagocytic engulfment.

Microglia continually surveil the brain allowing for rapid detection of tissue damage or infection. Microglial metabolism is linked to tissue homeostasis, yet how mitochondria are subcellularly partitioned in microglia and dynamically reorganize during surveillance, injury responses, and phagocytic engulfment in the intact brain are not known. Here, we performed intravital imaging of microglia mitochondria, revealing that microglial processes diverge, with some containing multiple mitochondria while others are completely void. Microglial processes that engage in minute-to-minute surveillance typically do not have mitochondria. Moreover, unlike process surveillance, mitochondrial motility does not change with animal anesthesia. Likewise, the processes that acutely chemoattract to a lesion site or initially engage with a neuron undergoing programmed cell death do not contain mitochondria. Rather, microglia mitochondria have a delayed arrival into the responding cell processes. Thus, there is subcellular heterogeneity of mitochondrial partitioning and asymmetry between mitochondrial localization and cell process motility or acute damage responses.

Crystal structures of cables formed by the acetylated and unacetylated forms of the Schizosaccharomyces pombe tropomyosin orthologue TpmCdc8

Cables formed by head-to-tail polymerization of tropomyosin, localized along the length of sarcomeric and cytoskeletal actin filaments, play a key role in regulating a wide range of motile and contractile processes. The stability of tropomyosin cables, their interaction with actin filaments and the functional properties of the resulting co-filaments are thought to be affected by N-terminal acetylation of tropomyosin. Here, we present high–resolution structures of cables formed by acetylated and unacetylated Schizosaccharomyces pombe tropomyosin orthologue TpmCdc8. The crystal structures represent different types of cables, each consisting of TpmCdc8 homodimers in a different conformation. The structures show how the interactions of the residues in the overlap junction contribute to cable formation and how local structural perturbations affect the conformational dynamics of the protein and its ability to transmit allosteric signals. In particular, N-terminal acetylation increases the helicity of the adjacent region, which leads to a local reduction in conformational dynamics and consequently to less fraying of the N-terminal region. This creates a more consistent complementary surface facilitating the formation of specific interactions across the overlap junction.

Motility-dependent processes in Toxoplasma gondii tachyzoites and bradyzoites: same same but different.

The tachyzoite stage of the apicomplexan parasite Toxoplasma gondii utilizes motility for multiple purposes during its lytic cycle, including host cell invasion, egress from infected cells, and migration to new uninfected host cells to repeat the process. Bradyzoite stage parasites, which establish a new infection in a naïve host, must also use motility to escape from the cysts that are ingested by the new host and then migrate to the gut wall, where they either invade cells of the intestinal epithelium or squeeze between these cells to infect the underlying connective tissue. We know very little about the motility of bradyzoites, which we analyze in detail here and compare to the well-characterized motility and motility-dependent processes of tachyzoites. Unexpectedly, bradyzoites were found to be as motile as tachyzoites in a 3D model extracellular matrix, and they showed increased invasion into and transmigration across certain cell types, consistent with their need to establish the infection in the gut. The motility of the two stages was inhibited to the same extent by cytochalasin D and KNX-002, compounds known to target the parasite's actomyosin-based motor. In contrast, other compounds that impact tachyzoite motility (tachyplegin and enhancer 5) have less of an effect on bradyzoites, and rapid bradyzoite egress from infected cells is not triggered by treatment with calcium ionophores, as it is with tachyzoites. The similarities and differences between these two life cycle stages highlight the need to characterize both tachyzoites and bradyzoites for a more complete understanding of the role of motility in the parasite life cycle and the effect that potential therapeutics targeting parasite motility will have on disease establishment and progression.

Orthologs of Plasmodium ICM1 are dispensable for Ca2+ mobilization in Toxoplasma gondii.

Apicomplexan parasites mobilize ionic calcium (Ca2+) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be important for calcium mobilization in Plasmodium falciparum and P. berghei. Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1_305470) and TgICM2-L (TGGT1_309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite's ability to mobilize Ca2+. These findings suggest that additional proteins may participate in Ca2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii. Collectively, these results highlight similar yet distinct mechanisms of Ca2+ mobilization between T. gondii and Plasmodium.IMPORTANCECa2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium, the necessity of ICM1 for Ca2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca2+ homeostasis and mobilization. Proteins involved in Ca2+ signaling in apicomplexans represent promising targets for therapeutic development.

The expression and clinical significance of CFAP65 in colon cancer

BackgroundCFAP65 (cilia and flagella associated protein 65) is a fundamental protein in the development and formation of ciliated flagella, but few studies have focused on its role in cancer. This study aimed to investigate the prognostic significance of CFAP65 in colon cancer.MethodsThe functionally enriched genes related to CFAP65 were analyzed through the Gene Ontology (GO) database. Subsequently, CFAP65 expression levels in colon cancer were evaluated by reverse transcription and quantitative polymerase chain reaction (RT-qPCR) and immunoblotting in 20 pairs of frozen samples, including tumors and their matched paratumor tissue. Furthermore, protein expression of CFAP65 in 189 colon cancer patients were assessed via immunohistochemical staining. The correlations between CFAP65 expression and clinical features as well as long-term survival were statistically analyzed.ResultsCFAP65-related genes are significantly enriched on cellular processes of cell motility, ion channels, and GTPase-associated signaling. The expression of CFAP65 was significantly higher in colon cancer tissue compared to paratumor tissue. The proportion of high expression and low expression of CFAP65 in the clinical samples of colon cancer were 61.9% and 38.1%, respectively, and its expression level was not associated with the clinical parameters including gender, age, tumor location, histological differentiation, tumor stage, vascular invasion and mismatch repair deficiency. The five-year disease-free survival rate of the patients with CFAP65 low expression tumors was significantly lower than that those with high expression tumors (56.9% vs. 72.6%, P = 0.03), but the overall survival rate has no significant difference (69% vs. 78.6%, P = 0.171). The cox hazard regression analysis model showed that CFAP65 expression, tumor stage and tumor location were independent prognostic factors.ConclusionsIn conclusion, we demonstrate CFAP65 is a potential predictive marker for tumor progression in colon cancer.

Antimicrobial and Antibiofilm Potential of Flourensia retinophylla against Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacteria with the greatest impact in the clinical area, due to the high rate of infections and deaths reaching every year. A previous scenario is associated with the bacteria's ability to develop resistance against conventional antibiotic therapies as well as biofilm formation. The above situation exhibits the necessity to reach new effective strategies against this pathogen. Flourensia retinophylla is a medicinal plant commonly used for bacterial infections treatments and has demonstrated antimicrobial effect, although its effect against S. aureus and bacterial biofilms has not been investigated. The purpose of this work was to analyze the antimicrobial and antibiofilm potential of F. retinophylla against S. aureus. The antimicrobial effect was determined using an ethanolic extract of F. retinophylla. The surface charge of the bacterial membrane, the K+ leakage and the effect on motility were determined. The ability to prevent and remove bacterial biofilms was analyzed in terms of bacterial biomass, metabolic activity and viability. The results showed that F. retinophylla presents inhibitory (MIC: 250 µg/mL) and bactericidal (MBC: 500 µg/mL) activity against S. aureus. The MIC extract increased the bacterial surface charge by 1.4 times and the K+ concentration in the extracellular medium by 60%. The MIC extract inhibited the motility process by 100%, 61% and 40% after 24, 48 and 72 h, respectively. The MIC extract prevented the formation of biofilms by more than 80% in terms of biomass production and metabolic activity. An extract at 10 × MIC reduced the metabolic activity by 82% and the viability by ≈50% in preformed biofilms. The results suggest that F. retinophylla affects S. areus membrane and the process of biofilm formation and removal. This effect could set a precedent to use this plant as alternative for antimicrobial and disinfectant therapies to control infections caused by this pathogen. In addition, this shrub could be considered for carrying out a purification process in order to identify the compounds responsible for the antimicrobial and antibiofilm effect.

Vimentin supports cell polarization by enhancing centrosome function and microtubule acetylation.

Cell polarity is important for controlling cell shape, motility and cell division processes. Vimentin intermediate filaments are important for cell migration and cell polarization in mesenchymal cells and assembly of vimentin and microtubule networks is dynamically coordinated, but the precise details of how vimentin mediates cell polarity remain unclear. Here, we characterize the effects of vimentin on the structure and function of the centrosome and the stability of microtubule filaments in wild-type and vimentin-null mouse embryonic fibroblasts. We find that vimentin mediates the structure of the pericentriolar material, promotes centrosome-mediated microtubule regrowth and increases the level of stable acetylated microtubules in the cell. Loss of vimentin also impairs centrosome repositioning during cell polarization and migration processes that occur during wound closure. Our results suggest that vimentin modulates centrosome structure and function as well as microtubule network stability, which has important implications for how cells establish proper cell polarization and persistent migration.

Abstract 4121: Unveiling discordant views of a novel drug's influence on cell motility from proteomics and immunofluorescence perspectives

Abstract Introduction: Metastasis is a leading cause of death in patients with solid tumors. Our group has discovered KBU2046, a selective inhibitor of cell motility (Nature Communications 2018). We conducted a time-course immunofluorescent staining to investigate the impact of KBU2046 on the cell adhesion and motility components of human prostate cancer cells. Our objective was to compare our findings with our proteomic analysis results in order to gain insights into the novel molecular mechanisms of KBU2046 in modulating cell motility. Experimental Procedures: Proteomics analysis. We treated PC3 cells with KBU2046 or vehicle (N=3/group) and quantified protein expression using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). In separate experiments (N=4), we performed LC-MS/MS after Tandem Mass Tag (TMT) labeling on isolated membrane fractions. We used bioinformatics tools, including Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway Analysis (IPA), and protein-protein interaction (PPI) network analysis, to characterize significantly differentially expressed proteins upon KBU2046 treatment and tried to verify by Western blots. Immunofluorescence (IF) staining. Following treatment of PC3, 1532CPTX, and 1532NPTX cells with KBU2046 or vehicle at various intervals, cells were stained for β1, β5 integrin, actin, and DAPI. Images were acquired by a Zeiss LSM 800 Confocal Laser Scanning Microscope. Results: The proteomics analysis demonstrated that KBU2046 was having a major impact on cell motility processes, consistent with our prior functional studies. Despite conducting Western blot analysis, we could not confirm the candidate proteins linked to cell adhesion. Using IF, we examined changes in cell adhesion proteins over time in response to KBU2046 treatment. The time-course IF investigation revealed that KBU2046 affects adhesion proteins kinetically by altering their subcellular localization over time rather than altering their total expression level. This finding corroborates KBU2046's known effects on cell motility, demonstrating that a kinetic-based analysis yields important new information not directly represented in a single time point screen, although functionally related. Conclusions: Our study underscores the importance of integrating proteomic and imaging methodologies to procure a holistic comprehension of the molecular mechanisms that govern cell behavior, especially those that relate to highly dynamic processes. Through these methodologies, we have obtained a more profound understanding of how cells regulate motility and how that process can be therapeutically modulated. Citation Format: Weining Chen, Nicholas Woods, Henry Chun Hin Law, Fangfang Qiao, Sankarasubramanian Jagadesan, Chittibabu Guda, Raymond Bergan. Unveiling discordant views of a novel drug's influence on cell motility from proteomics and immunofluorescence perspectives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4121.

APOE4 genotype and aging impair injury-induced microglial behavior in brain slices, including toward Aβ, through P2RY12

Microglia are highly dynamic cells that play a critical role in tissue homeostasis through the surveillance of brain parenchyma and response to cues associated with damage. Aging and APOE4 genotype are the strongest risk factors for Alzheimer’s disease (AD), but how they affect microglial dynamics remains unclear. Using ex vivo confocal microscopy, we analyzed microglial dynamic behaviors in the entorhinal cortex (EC) and hippocampus CA1 of 6-, 12-, and 21-month-old mice APOE3 or APOE4 knock-in mice expressing GFP under the CX3CR1 promoter. To study microglia surveillance, we imaged microglia baseline motility for 20 min and measured the extension and retraction of processes. We found that APOE4 microglia exhibited significantly less brain surveillance (27%) compared to APOE3 microglia in 6-month-old mice; aging exacerbated this deficit. To measure microglia response to damage, we imaged process motility in response to ATP, an injury-associated signal, for 30 min. We found APOE4 microglia extended their processes significantly slower (0.9 µm/min, p < 0.005) than APOE3 microglia (1.1 μm/min) in 6-month-old animals. APOE-associated alterations in microglia motility were observed in 12- and 21-month-old animals, and this effect was exacerbated with aging in APOE4 microglia. We measured protein and mRNA levels of P2RY12, a core microglial receptor required for process movement in response to damage. We found that APOE4 microglia express significantly less P2RY12 receptors compared to APOE3 microglia despite no changes in P2RY12 transcripts. To examine if the effect of APOE4 on the microglial response to ATP also applied to amyloid β (Aβ), we infused locally Hi-Lyte Fluor 555-labeled Aβ in acute brain slices of 6-month-old mice and imaged microglia movement for 2 h. APOE4 microglia showed a significantly slower (p < 0.0001) process movement toward the Aβ, and less Aβ coverage at early time points after Aβ injection. To test whether P2RY12 is involved in process movement in response to Aβ, we treated acute brain slices with a P2RY12 antagonist before Aβ injection; microglial processes no longer migrated towards Aβ. These results provide mechanistic insights into the impact of APOE4 genotype and aging in dynamic microglial behaviors prior to gross Aβ pathology and could help explain how APOE4 brains are more susceptible to AD pathogenesis.

Bio-Inspired Magnetically Responsive Silicone Cilia: Fabrication Strategy and Interaction with Biological Mucus.

Cilia are biological structures essential to drive the mobility of secretions and maintain the proper function of the respiratory airways. However, this motile self-cleaning process is significantly compromised in the presence of silicone tracheal prosthesis, leading to biofilm growth and impeding effective treatment. To address this challenge and enhance the performance of these devices, we propose the fabrication of magnetic silicone cilia, with the prospect of their integration onto silicone prostheses. The present study presents a fabrication method based on magnetic self-assembly and assesses the interaction behavior of the cilia array with biological mucus. This protocol allows for the customization of cilia dimensions across a wide range of aspect ratios (from 6 to 85) and array densities (from 10 to 80 cilia/mm2) by adjusting the fabrication parameters, offering flexibility for adjustments according to their required characteristics. Furthermore, we evaluated the suitability of different cilia arrays for biomedical applications by analyzing their interaction with bullfrog mucus, simulating the airways environment. Our findings demonstrate that the fabricated cilia are mechanically resistant to the viscous fluid and still exhibit controlled movement under the influence of an external moving magnet. A correlation between cilia dimensions and mucus wettability profile suggests a potential role in facilitating mucus depuration, paving the way for further advancements aimed at enhancing the performance of silicone prostheses in clinical settings.

The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions.

Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.

Structural analysis of PTPN21 reveals a dominant-negative effect of the FERM domain on its phosphatase activity.

PTPN21 belongs to the four-point-one, ezrin, radixin, moesin (FERM) domain-containing protein tyrosine phosphatases (PTP) and plays important roles in cytoskeleton-associated cellular processes like cell adhesion, motility, and cargo transport. Because of the presence of a WPE loop instead of a WPD loop in the phosphatase domain, it is often considered to lack phosphatase activity. However, many of PTPN21's biological functions require its catalytic activity. To reconcile these findings, we have determined the structures of individual PTPN21 FERM, PTP domains, and a complex between FERM-PTP. Combined with biochemical analysis, we have found that PTPN21 PTP is weakly active and is autoinhibited by association with its FERM domain. Disruption of FERM-PTP interaction results in enhanced ERK activation. The oncogenic HPV18 E7 protein binds to PTP at the same location as PTPN21 FERM, indicating that it may act by displacing the FERM domain from PTP. Our results provide mechanistic insight into PTPN21 and benefit functional studies of PTPN21-mediated processes.

Structures and diversities of bacterial communities in oil-contaminated soil at shale gas well site assessed by high-throughput sequencing.

Currently, there is limited understanding of the structures and variabilities of bacterial communities in oil-contaminated soil within shale gas development. The Changning shale gas well site in Sichuan province was focused, and high-throughput sequencing was used to investigate the structures of bacterial communities and functions of bacteria in soil with different degrees of oil pollution. Furthermore, the influences of the environmental factors including pH, moisture content, organic matter, total nitrogen, total phosphorus, oil, and the biological toxicity of the soil on the structures of bacterial communities were analyzed. The results revealed that Proteobacteria and Firmicutes predominated in the oil-contaminated soil. α-Proteobacteria and γ-Proteobacteria were the main classes under the Proteobacteria phylum. Bacilli was the main class in the Firmicutes phylum. Notably, more bacteria were only found in CN-5 which was the soil near the storage pond for abandoned drilling mud, including Marinobacter, Balneola, Novispirillum, Castellaniella, and Alishewanella. These bacteria exhibited resilience to higher toxicity and demonstrated proficiency in oil degradation. The functions including carbohydrate transport and metabolism, energy metabolism, replication, recombination and repair replication, signal transduction mechanisms, and amino acid transport and metabolism responded differently to varying concentrations of oil. The disparities in bacterial genus composition across samples stemmed from a complex play of pH, moisture content, organic matter, total nitrogen, total phosphorus, oil concentration, and biological toxicity. Notably, bacterial richness correlated positively with moisture content, while bacterial diversity showed a significant positive correlation with pH. Acidobacteria exhibited a significant positive correlation with moisture content. Litorivivens and Luteimonas displayed a significant negative correlation with pH, while Rhizobium exhibited a significant negative correlation with moisture content. Pseudomonas, Proteiniphilum, and Halomonas exhibited positive correlations not only with organic matter but also with oil concentration. Total nitrogen exhibited a significant positive correlation with Taonella and Sideroxydans. On the other hand, total phosphorus showed a significant negative correlation with Sphingomonas. Furthermore, Sphingomonas, Gp6, and Ramlibacter displayed significant negative correlations with biological toxicity. The differential functions exhibited no significant correlation with environmental factors but displayed a significant positive correlation with the Proteobacteria phylum. Aridibacter demonstrated a significant positive correlation with cell motility and cellular processes and signaling. Conversely, Pseudomonas, Proteiniphilum, and Halomonas were negatively correlated with differential functions, particularly in amino acid metabolism, carbohydrate metabolism, and membrane transport. Compared with previous research, more factors were considered in this research when studying structural changes in bacterial communities, such as physicochemical properties and biological toxicity of soil. In addition, the correlations of differential functions of communities with environmental factors, bacterial phyla, and genera were investigated.

Synaptic vesicle release regulates pre-myelinating oligodendrocyte-axon interactions in a neuron subtype-specific manner.

Oligodendrocyte-lineage cells are central nervous system (CNS) glia that perform multiple functions including the selective myelination of some but not all axons. During myelination, synaptic vesicle release from axons promotes sheath stabilization and growth on a subset of neuron subtypes. In comparison, it is unknown if pre-myelinating oligodendrocyte process extensions selectively interact with specific neural circuits or axon subtypes, and whether the formation and stabilization of these neuron-glia interactions involves synaptic vesicle release. In this study, we used fluorescent reporters in the larval zebrafish model to track pre-myelinating oligodendrocyte process extensions interacting with spinal axons utilizing in vivo imaging. Monitoring motile oligodendrocyte processes and their interactions with individually labeled axons revealed that synaptic vesicle release regulates the behavior of subsets of process extensions. Specifically, blocking synaptic vesicle release decreased the longevity of oligodendrocyte process extensions interacting with reticulospinal axons. Furthermore, blocking synaptic vesicle release increased the frequency that new interactions formed and retracted. In contrast, tracking the movements of all process extensions of singly-labeled oligodendrocytes revealed that synaptic vesicle release does not regulate overall process motility or exploratory behavior. Blocking synaptic vesicle release influenced the density of oligodendrocyte process extensions interacting with reticulospinal and serotonergic axons, but not commissural interneuron or dopaminergic axons. Taken together, these data indicate that alterations to synaptic vesicle release cause changes to oligodendrocyte-axon interactions that are neuron subtype specific.

A passive star polymer in a dense active bath: insights from computer simulations.

Using computer simulations in two dimensions (2D), we explore the structure and dynamics of a star polymer with three arms made of passive monomers immersed in a bath of active Brownian particles (ABPs). We analyze the conformational and dynamical changes of the polymer as a function of activity and packing fraction. We also study the process of motility induced phase separation (MIPS) in the presence of a star polymer, which acts as a mobile nucleation center. The presence of the polymer increases the growth rate of the clusters in comparison to a bath without the polymer. In particular, for low packing fraction, both nucleation and cluster growth are affected by the inclusion of the star polymer. Clusters grow in the vicinity of the star polymer, resulting in the star polymer experiencing a caged motion similar to a tagged ABP in the dense phase. Due to the topological constraints of the star polymers and clustering nearby, the conformational changes of the star polymer lead to interesting observations. Inter alia, we observe the shrinking of the arm with increasing activity along with a short-lived hairpin structure of one arm formed. We also see the transient pairing of two arms of the star polymer, while the third is largely separated at high activity. We hope our findings will help in understanding the behavior of active-passive mixtures, including biopolymers of complex topology in dense active suspensions.

Synapse Regulation.

Microglia are the resident immune cells of the brain. As such, they rapidly detect changes in normal brain homeostasis and accurately respond by fine-tuning in a tightly regulated manner their morphology, gene expression, and functional behavior. Depending on the nature of these changes, microglia can thicken and retract their processes, proliferate and migrate, release numerous signaling factors and compounds influencing neuronal physiology (e.g., cytokines and trophic factors), in addition to secreting proteases able to transform the extracellular matrix, and phagocytosing various types of cellular debris, etc. Because microglia also transform rapidly (on a time scale of minutes) during experimental procedures, studying these very special cells requires methods that are specifically non-invasive. The development of such methods has provided unprecedented insights into the roles of microglia during normal physiological conditions. In particular, transcranial two-photon in vivo imaging revealed that presumably "resting" microglia continuously survey the brain parenchyma with their highly motile processes, in addition to modulating their structural and functional interactions with neuronal circuits along the changes in neuronal activity and behavioral experience occurring throughout the lifespan. In this chapter, we will describe how surveillant microglia interact with synaptic elements and modulate the number, maturation, function, and plasticity of synapses in the healthy developing, mature, and aging brain, with consequences on neuronal activity, learning and memory, and the behavioral outcome.

Direct observation of cortactin protecting Arp2/3-actin filament branch junctions from GMF-mediated destabilization

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF’s branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin’s on-rate (3 ×107 s−1 M−1) and off-rate (0.03 s−1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

Subcellular mechano-regulation of cell migration in confined extracellular microenvironment.

Cell migration is a highly coordinated cellular event that determines diverse physiological and pathological processes in which the continuous interaction of a migrating cell with neighboring cells or the extracellular matrix is regulated by the physical setting of the extracellular microenvironment. In confined spaces, cell migration occurs differently compared to unconfined open spaces owing to the additional forces that limit cell motility, which create a driving bias for cells to invade the confined space, resulting in a distinct cell motility process compared to what is expected in open spaces. Moreover, cells in confined environments can be subjected to elevated mechanical compression, which causes physical stimuli and activates the damage repair cycle in the cell, including the DNA in the nucleus. Although cells have a self-restoring system to repair damage from the cell membrane to the genetic components of the nucleus, this process may result in genetic and/or epigenetic alterations that can increase the risk of the progression of diverse diseases, such as cancer and immune disorders. Furthermore, there has been a shift in the paradigm of bioengineering from the development of new biomaterials to controlling biophysical cues and fine-tuning cell behaviors to cure damaged/diseased tissues. The external physical cues perceived by cells are transduced along the mechanosensitive machinery, which is further channeled into the nucleus through subcellular molecular linkages of the nucleoskeleton and cytoskeleton or the biochemical translocation of transcription factors. Thus, external cues can directly or indirectly regulate genetic transcriptional processes and nuclear mechanics, ultimately determining cell fate. In this review, we discuss the importance of the biophysical cues, response mechanisms, and mechanical models of cell migration in confined environments. We also discuss the effect of force-dependent deformation of subcellular components, specifically focusing on subnuclear organelles, such as nuclear membranes and chromosomal organization. This review will provide a biophysical perspective on cancer progression and metastasis as well as abnormal cellular proliferation.

GFAP Upregulation by Astrocytes May Attenuate Phospho‐Tau Levels in a Tauopathy Mouse Model

AbstractBackgroundAstrocytes react to amyloid‐β plaques and phospho‐tau (pTau) neurofibrillary tangles in the Alzheimer’s disease brain but their net effect on these Alzheimer’s neuropathological hallmarks remains controversial. A well‐known feature of these so‐called “reactive astrocytes” is the upregulation of the intermediate filament glial fibrillary acidic protein (GFAP), which is thought to be necessary for astrocyte process motility and glial scar formation. Here we asked whether this cytoskeletal remodeling helps reactive astrocytes control the burden of pTau species and/or protect synapses and neurons.MethodWe overexpressed human GFAP isoform 1 specifically in astrocytes of 4‐month‐old, sex‐balanced, THY‐Tau22 mice (Thy1.2‐MAPT G272V/P301S 1N4R, GFAP mice, n = 11)—when neurofibrillary tangles start to appear in the hippocampal CA1 subfield—using a viral transfer strategy with single retro‐orbital intravenous injection of AAV‐PHP.B (2.5×1011 vg) and the gfaABC1D promoter. We euthanized the mice at 11 months of age (i.e., prior to the tauopathy plateau) and performed immunohistochemical and/or biochemical analysis of astrocyte, tau, and synaptic markers. Negative control groups consisted of THY‐Tau22 littermates injected with either phosphate‐buffered saline (PBS mice, n = 9) or an AAV‐PHP.B vector encoding the enhanced green fluorescent protein (EGFP mice, n = 10).ResultsImmunohistochemistry confirmed the astrocyte‐specific expression of human GFAP with an apparent integration in their intermediate filament network. We observed statistically significant reductions or marginally significant trends toward decrease in hippocampal SDS‐soluble levels AT8/pTauSer202/Thr205 and AT8‐immunoreactive area fraction, and a shift of AT180/pTauThr231 from SDS‐soluble (decreased) to TBS‐soluble (increased) hippocampal protein fractions, in GFAP vs. EGFP and/or PBS mice, but no changes in soluble or insoluble total tau or AT270/pTauThr181 across groups. In the cortex, we detected no changes in soluble or insoluble total tau, AT8/pTauSer202/Thr205, or AT270/pTauThr181 levels, nor in AT8‐immunoreactive area. Lastly, we found no significant differences across groups in brain weight, cortical or hippocampal areas, nor in the cortical levels of synaptophysin, Psd95, or the glutamate transporters Eaat1/Glast‐1 and Eaat2/Glt‐1.ConclusionOur findings indicate that GFAP upregulation by astrocytes—a classic feature of reactive astrogliosis—may attenuate neuronal pTau burden in a tauopathy mouse model. Ongoing work will further elucidate whether this aspect of reactive astrocytes impacts pTau‐induced neurodegeneration.

Cortical microglia dynamics are conserved during voluntary wheel running.

We present the first demonstration of chronic in vivo imaging of microglia in mice undergoing voluntary wheel running. We find that healthy mice undergoing voluntary wheel running have similar microglia dynamics, morphologies, and responses to injury when compared to sedentary mice. This suggests that exercise over a period of 1 mo does not grossly alter cortical microglial phenotypes and that exercise may exert its beneficial effects on the brain through other mechanisms. Future work examining how microglia dynamics may be altered during exercise in disease or injury models could provide further insights into the therapeutic benefit of exercise.NEW & NOTEWORTHY We demonstrate the first use of chronic in vivo imaging of microglia over time during physical exercise. We found that microglia movement, morphology, and process motility were remarkably stable during voluntary wheel running (VWR). Additionally, microglia in running mice respond similarly to laser ablation injury compared to sedentary mice. These findings indicate that VWR does not induce changes in microglia dynamics in healthy adults. Exercise may elicit positive effects on the brain through other mechanisms.