Dictyostelium Discoideum Research Articles

Simple to Complex: The Role of Actin and Microtubules in Mitochondrial Dynamics in Amoeba, Yeast, and Mammalian Cells.

Mitochondria are complex organelles that provide energy for the cell in the form of adenosine triphosphate (ATP) and have very specific structures. For most organisms, this is a reticular or tubular mitochondrial network, while others have singular oval-shaped organelles. Nonetheless, maintenance of this structure is dependent on the mitochondrial dynamics, fission, fusion, and motility. Recently, studies have shown that the cytoskeleton has a significant role in the regulation of mitochondrial dynamics. In this review, we focus on microtubules and actin filaments and look at what is currently known about the cytoskeleton’s role in mitochondrial dynamics in complex models like mammals and yeast, as well as what is known in the simple model system, Dictyostelium discoideum. Understanding how the cytoskeleton is involved in mitochondrial dynamics increases our understanding of mitochondrial disease, especially neurodegenerative diseases. Increases in fission, loss of fusion, and fragmented mitochondria are seen in several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s disease. There is no known cure for these diseases, but new therapeutic strategies using drugs to alter mitochondrial fusion and fission activity are being considered. The future of these therapeutic studies is dependent on an in-depth understanding of the mechanisms of mitochondrial dynamics. Understanding the cytoskeleton’s role in dynamics in multiple model organisms will further our understanding of these mechanisms and could potentially uncover new therapeutic targets for these neurodegenerative diseases.

Oxygen-dependent regulation of E3(SCF)ubiquitin ligases and a Skp1-associated JmjD6 homolog in development of the social amoeba Dictyostelium

E3-SCF (Skp1/cullin-1/F-box protein) polyubiquitin ligases activate the proteasomal degradation of over a thousand proteins, but the evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged their elucidation in protists. Here, we expand the FBP candidate list in the social amoeba Dictyostelium and show that the Skp1 interactome is highly remodeled as cells transition from growth to multicellular development. Importantly, a subset of candidate FBPs was less represented when the posttranslational hydroxylation and glycosylation of Skp1 was abrogated by deletion of the O2-sensing Skp1 prolyl hydroxylase PhyA. A role for this Skp1 modification for SCF activity was indicated by partial rescue of development, which normally depends on high O2 and PhyA, of phyA-KO cells by proteasomal inhibitors. Further examination of two FBPs, FbxwD and the Jumonji C protein JcdI, suggested that Skp1 was substituted by other factors in phyA-KO cells. Although a double-KO of jcdI and its paralog jcdH did not affect development, overexpression of JcdI increased its sensitivity to O2. JcdI, a nonheme dioxygenase shown to have physiological O2 dependence, is conserved across protists with its F-box and other domains, and is related to the human oncogene JmjD6. Sensitization of JcdI-overexpression cells to O2 depended on its dioxygenase activity and other domains, but not its F-box, which may however be the mediator of its reduced levels in WT relative to Skp1 modification mutant cells. The findings suggest that activation of JcdI by O2 is tempered by homeostatic downregulation via PhyA and association with Skp1.

Host–Endosymbiont Relationship Impacts the Retention of Bacteria-Containing Amoeba Spores in Porous Media

Amoebae are protists that are commonly found in water, soil, and other habitats around the world and have complex interactions with other microorganisms. In this work, we investigated how host-endosymbiont interactions between amoebae and bacteria impacted the retention behavior of amoeba spores in porous media. A model amoeba species, Dictyostelium discoideum, and a representative bacterium, Burkholderia agricolaris B1qs70, were used to prepare amoeba spores that carried bacteria. After interacting with B. agricolaris, the retention of D. discoideum spores was enhanced compared to noninfected spores. Diverse proteins, especially proteins contributing to the looser exosporium structure and cell adhesion functionality, are secreted in higher quantities on the exosporium surface of infected spores compared to that of noninfected ones. Comprehensive examinations using a quartz crystal microbalance with dissipation (QCM-D), a parallel plate chamber, and a single-cell force microscope present coherent evidence that changes in the exosporium of D. discoideum spores due to infection by B. agricolaris enhance the connections between spores in the suspension and the spores that were previously deposited on the collector surface, thus resulting in more retention compared to the uninfected ones in porous media. This work provides novel insight into the retention of amoeba spores after bacterial infection in porous media and suggests that the host-endosymbiont relationship regulates the fate of biocolloids in drinking water systems, groundwater, and other porous environments.

Calcium responses to external mechanical stimuli in the multicellular stage of Dictyostelium discoideum

Calcium acts as a second messenger to regulate many cellular functions, including cell motility. In Dictyostelium discoideum, the cytosolic calcium level oscillates synchronously, and calcium waves propagate through the cell population during the early stages of development, including aggregation. In the unicellular phase, the calcium response through Piezo channels also functions in mechanosensing. However, calcium dynamics during multicellular morphogenesis are still unclear. Here, live imaging of cytosolic calcium revealed that calcium wave propagation, depending on cAMP relay, disappeared at the onset of multicellular body (slug) formation. Later, other forms of occasional calcium bursts and their propagation were observed in both anterior and posterior regions of migrating slugs. This calcium signaling also occurred in response to mechanical stimuli. Two pathways—calcium release from the endoplasmic reticulum via IP3 receptor and calcium influx from outside the cell—were involved in calcium signals induced by mechanical stimuli. These data suggest that calcium signaling is involved in mechanosensing in both the unicellular and multicellular phases of Dictyostelium development using different molecular mechanisms.

The Polarized Redistribution of the Contractile Vacuole to the Rear of the Cell is Critical for Streaming and is Regulated by PI(4,5)P2-Mediated Exocytosis

Dictyostelium discoideum amoebae align in a head to tail manner during the process of streaming during fruiting body formation. The chemoattractant cAMP is the chemoattractant regulating cell migration during this process and is released from the rear of cells. The process by which this cAMP release occurs has eluded investigators for many decades, but new findings suggest that this release can occur through expulsion during contractile vacuole (CV) ejection. The CV is an organelle that performs several functions inside the cell including the regulation of osmolarity, and discharges its content via exocytosis. The CV localizes to the rear of the cell and appears to be part of the polarity network, with the localization under the influence of the plasma membrane (PM) lipids, including the phosphoinositides (PIs), among those is PI(4,5)P2, the most abundant PI on the PM. Research on D. discoideum and neutrophils have shown that PI(4,5)P2 is enriched at the rear of migrating cells. In several systems, it has been shown that the essential regulator of exocytosis is through the exocyst complex, mediated in part by PI(4,5)P2-binding. This review features the role of the CV complex in D. discoideum signaling with a focus on the role of PI(4,5)P2 in regulating CV exocytosis and localization. Many of the regulators of these processes are conserved during evolution, so the mechanisms controlling exocytosis and membrane trafficking in D. discoideum and mammalian cells will be discussed, highlighting their important functions in membrane trafficking and signaling in health and disease.

Sex-Based Differences in Human Neutrophil Chemorepulsion.

A considerable amount is known about how eukaryotic cells move toward an attractant, and the mechanisms are conserved from Dictyostelium discoideum to human neutrophils. Relatively little is known about chemorepulsion, where cells move away from a repellent signal. We previously identified pathways mediating chemorepulsion in Dictyostelium, and here we show that these pathways, including Ras, Rac, protein kinase C, PTEN, and ERK1 and 2, are required for human neutrophil chemorepulsion, and, as with Dictyostelium chemorepulsion, PI3K and phospholipase C are not necessary, suggesting that eukaryotic chemorepulsion mechanisms are conserved. Surprisingly, there were differences between male and female neutrophils. Inhibition of Rho-associated kinases or Cdc42 caused male neutrophils to be more repelled by a chemorepellent and female neutrophils to be attracted to the chemorepellent. In the presence of a chemorepellent, compared with male neutrophils, female neutrophils showed a reduced percentage of repelled neutrophils, greater persistence of movement, more adhesion, less accumulation of PI(3,4,5)P3, and less polymerization of actin. Five proteins associated with chemorepulsion pathways are differentially abundant, with three of the five showing sex dimorphism in protein localization in unstimulated male and female neutrophils. Together, this indicates a fundamental difference in a motility mechanism in the innate immune system in men and women.

Developmental constraints enforce altruism and avert the tragedy of the commons in a social microbe

Organisms often cooperate through the production of freely available public goods. This can greatly benefit the group but is vulnerable to the "tragedy of the commons" if individuals lack the motivation to make the necessary investment into public goods production. Relatedness to groupmates can motivate individual investment because group success ultimately benefits their genes' own self-interests. However, systems often lack mechanisms that can reliably ensure that relatedness is high enough to promote cooperation. Consequently, groups face a persistent threat from the tragedy unless they have a mechanism to enforce investment when relatedness fails to provide adequate motivation. To understand the real threat posed by the tragedy and whether groups can avert its impact, we determine how the social amoeba Dictyostelium discoideum responds as relatedness decreases to levels that should induce the tragedy. We find that, while investment in public goods declines as overall within-group relatedness declines, groups avert the expected catastrophic collapse of the commons by continuing to invest, even when relatedness should be too low to incentivize any contribution. We show that this is due to a developmental buffering system that generates enforcement because insufficient cooperation perturbs the balance of a negative feedback system controlling multicellular development. This developmental constraint enforces investment under the conditions expected to be most tragic, allowing groups to avert a collapse in cooperation. These results help explain how mechanisms that suppress selfishness and enforce cooperation can arise inadvertently as a by-product of constraints imposed by selection on different traits.

Novel Sterically Crowded and Conformationally Constrained α-Aminophosphonates with a Near-Neutral pKa as Highly Accurate 31P NMR pH Probes. Application to Subtle pH Gradients Determination in Dictyostelium discoideum Cells.

In order to discover new 31P NMR markers for probing subtle pH changes (<0.2 pH unit) in biological environments, fifteen new conformationally constrained or sterically hindered α-aminophosphonates derived from diethyl(2-methylpyrrolidin-2-yl)phosphonate were synthesized and tested for their pH reporting and cytotoxic properties in vitro. All compounds showed near-neutral pKas (ranging 6.28–6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δδab between the acidic and basic forms) ranging from 9.2–10.7 ppm, being fourfold larger than conventional endogenous markers such as inorganic phosphate. X-ray crystallographic studies combined with predictive empirical relationships and ab initio calculations addressed the inductive and stereochemical effects of substituents linked to the protonated amine function. Satisfactory correlations were established between pKas and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δδab. Finally, the hit 31P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing pKa = 6.45 with Δδab = 10.64 ppm, allowed the first clear-cut evidence of trans-sarcolemmal pH gradients in normoxic Dictyostelium discoideum cells with an accuracy of <0.05 pH units.

CRISPR/Cas9-based genome-wide screening of Dictyostelium

Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a unique life cycle and is often used as a crucial research model for a wide range of biological processes and rare metabolites. To address the inadequacies of conventional genetic screening approaches, we developed a highly efficient CRISPR/Cas9-based genome-wide screening system for Dictyostelium. A genome-wide library of 27,405 gRNAs and a kinase library of 4,582 gRNAs were compiled and mutant pools were generated. The resulting mutants were screened for defects in cell growth and more than 10 candidate genes were identified. Six of these were validated and five recreated mutants presented with growth abnormalities. Finally, the genes implicated in developmental defects were screened to identify the unknown genes associated with a phenotype of interest. These findings demonstrate the potential of the CRISPR/Cas9 system as an efficient genome-wide screening method.

Insights on Microsatellite Characteristics, Evolution, and Function From the Social Amoeba Dictyostelium discoideum.

Microsatellites are repetitive sequences commonly found in the genomes of higher organisms. These repetitive sequences are prone to expansion or contraction, and when microsatellite expansion occurs in the regulatory or coding regions of genes this can result in a number of diseases including many neurodegenerative diseases. Unlike in humans and other organisms, the social amoeba Dictyostelium discoideum contains an unusually high number of microsatellites. Intriguingly, many of these microsatellites fall within the coding region of genes, resulting in nearly 10,000 homopolymeric repeat proteins within the Dictyostelium proteome. Surprisingly, among the most common of these repeats are polyglutamine repeats, a type of repeat that causes a class of nine neurodegenerative diseases in humans. In this minireview, we summarize what is currently known about homopolymeric repeats and microsatellites in Dictyostelium discoideum and discuss the potential utility of Dictyostelium for identifying novel mechanisms that utilize and regulate regions of repetitive DNA.

Mfsd8 Modulates Growth and the Early Stages of Multicellular Development in Dictyostelium discoideum.

MFSD8 is a transmembrane protein that has been reported to transport chloride ions across the lysosomal membrane. Mutations in MFSD8 are associated with a subtype of Batten disease called CLN7 disease. Batten disease encompasses a family of 13 inherited neurodegenerative lysosomal storage diseases collectively referred to as the neuronal ceroid lipofuscinoses (NCLs). Previous work identified an ortholog of human MFSD8 in the social amoeba D. discoideum (gene: mfsd8, protein: Mfsd8), reported its localization to endocytic compartments, and demonstrated its involvement in protein secretion. In this study, we further characterized the effects of mfsd8 loss during D. discoideum growth and early stages of multicellular development. During growth, mfsd8 − cells displayed increased rates of proliferation, pinocytosis, and expansion on bacterial lawns. Loss of mfsd8 also increased cell size, inhibited cytokinesis, affected the intracellular and extracellular levels of the quorum-sensing protein autocrine proliferation repressor A, and altered lysosomal enzyme activity. During the early stages of development, loss of mfsd8 delayed aggregation, which we determined was at least partly due to impaired cell-substrate adhesion, defects in protein secretion, and alterations in lysosomal enzyme activity. Overall, these results show that Mfsd8 plays an important role in modulating a variety of processes during the growth and early development of D. discoideum.

Context dependence in the symbiosis between Dictyostelium discoideum and Paraburkholderia.

Symbiotic interactions change with environmental context. Measuring these context‐dependent effects in hosts and symbionts is critical to determining the nature of symbiotic interactions. We investigated context dependence in the symbiosis between social amoeba hosts and their inedible Paraburkholderia bacterial symbionts, where the context is the abundance of host food bacteria. Paraburkholderia have been shown to harm hosts dispersed to food‐rich environments, but aid hosts dispersed to food‐poor environments by allowing hosts to carry food bacteria. Through measuring symbiont density and host spore production, we show that this food context matters in three other ways. First, it matters for symbionts, who suffer a greater cost from competition with food bacteria in the food‐rich context. Second, it matters for host‐symbiont conflict, changing how symbiont density negatively impacts host spore production. Third, data‐based simulations show that symbiosis often provides a long‐term fitness advantage for hosts after rounds of growth and dispersal in variable food contexts, especially when conditions are harsh with little food. These results show how food context can have many consequences for the Dictyostelium‐Paraburkholderia symbiosis and that both sides can frequently benefit.

IDENTIFICATION OF WEE GENES IN DICTYOSTELIUM DISCOIDEUM

Cell cycle is a basic cellular mechanism that allows for cellular growth and development, regeneration or repair, and wear of tissues or even regeneration of the whole organism in certain cases. A failure during the cell cycle process leads to various dire consequences. Various proteins and kinases play a vital role in the regulation of the cell cycle to meet cellular demands and needs. Amongst the kinases, Wee1 is a key regulator of cell cycle progression which inhibits mitotic entry by phosphorylating the tyrosine residue on the M-phase-inducing kinase, Cdk1, at the G2/M transition. In Dictyostelium discoideum, wee1 genes that regulate the cell cycle progression at the G2/M transition have not been identified and studied yet. Our findings support the existence of the Wee1 homolog in D. discoideum, which could play a role in cell division at G2/M.

Centrosome Positioning in Migrating Dictyostelium Cells.

Directional cell migration and the establishment of polarity play an important role in development, wound healing, and host cell defense. While actin polymerization provides the driving force at the cell front, the microtubule network assumes a regulatory function, in coordinating front protrusion and rear retraction. By using Dictyostelium discoideum cells as a model for amoeboid movement in different 2D and 3D environments, the position of the centrosome relative to the nucleus was analyzed using live-cell microscopy. Our results showed that the centrosome was preferentially located rearward of the nucleus under all conditions tested for directed migration, while the nucleus was oriented toward the expanding front. When cells are hindered from straight movement by obstacles, the centrosome is displaced temporarily from its rearward location to the side of the nucleus, but is reoriented within seconds. This relocalization is supported by the presence of intact microtubules and their contact with the cortex. The data suggest that the centrosome is responsible for coordinating microtubules with respect to the nucleus. In summary, we have analyzed the orientation of the centrosome during different modes of migration in an amoeboid model and present evidence that the basic principles of centrosome positioning and movement are conserved between Dictyostelium and human leukocytes.

Cells responding to chemoattractant on a structured substrate

Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.

Soil Amoebae Affect Iron and Chromium Reduction through Preferential Predation between Two Metal-Reducing Bacteria.

Soil protists are essential but often overlooked in soil and could impact microbially driven element cycling in natural ecosystems. However, how protists influence heavy metal cycling in soil remains poorly understood. In this study, we used a model protist, Dictyostelium discoideum, to explore the effect of interactions between soil amoeba and metal-reducing bacteria on the reduction of soil Fe(III) and Cr(VI). We found that D. discoideum could preferentially prey on the Fe(III)-reducing bacterium Shewanella decolorationis S12 and significantly decrease its biomass. Surprisingly, this predation pressure also stimulated the activity of a single S. decolorationis S12 bacterium to reduce Fe(III) by enhancing the content of electron-transfer protein cyt c, intracellular ATP synthesis, and reactive oxygen species (e.g., H2O2). We also found that D. discoideum could not prey on the Cr(VI)-reducing bacterium Brevibacillus laterosporus. In contrast, B. laterosporus became edible to amoebae in the presence of S. decolorationis S12, and their Cr(VI) reduction ability decreased under amoeba predation pressure. This study provides direct evidence that protists can affect the Cr and Fe cycling via the elective predation pressure on the metal-reducing bacteria, broadening our horizons of predation of protists on soil metal cycling.

Using Dictyostelium discoideum as a model for phagocytosis resistance in Escherichia coli

Abstract Escherichia coli related illnesses continue to be a problem around the world, especially in developing countries that lack clean water and proper sanitation. Dictyostelium discoideum is a soil dwelling amoeba that phagocytizes bacteria for nutrients. D. discoideum is a great model to study host pathogen interactions, and it has been used to identify virulence factors in bacteria. Our objective is to use Dictyostelium discoideum as a model to identify bacterial genes involved in resistance to phagocytosis. We hypothesize that D. discoideum can be used as a model organism to identify virulence genes associated with E. coli. The aims of this study are to identify E. coli genes predicted to grant resistance to D. discoideum grazing and phagocytosis and further characterize genes identified as potential virulence factors and observe the genes’ effects on D. discoideum grazing and phagocytic killing. Intestinal pathogenic, extraintestinal pathogenic, and laboratory E. coli strains were classified according to grazing phenotype. Previous comparative genomic analysis by large-scale BLAST score ratio (LS-BSR) identified genes prevalent amongst strains resistant to D. discoideum grazing. The gene gipA (growth in Peyer’s patches) was one gene found in various E. coli strains resistant to D. discoideum grazing. We are currently working to characterize the role of E. coli genes associated with resistance to D. discoideum grazing by manipulating E. coli strains for expression of these genes. We will observe the interactions between these strains and D. discoideum grazing and phagocytosis. This experiment will give insight into bacterial genes and virulence factors responsible for evading phagocytosis and the immune system. This project was funded in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institute of Health, and Department of Health and Human Services under grant number U19 AI110820. Dr. M. Snyder was funded by an NIH NIGMS grant F33 GM116618, a Franklin Research Grant from the American Philosophical Society and a grant from the TU FDRC committee. This research was also supported by an NIH NIGMS R25 GM19970.

Role of Adhesion Mediator SibA in Mechanotransduction of Dictyostelium discoideum

Dictyostelium discoideum is a social amoeba commonly used as a model organism for the study of various cellular processes, including directed cell migration. Mechanical cues, such as shear flow, have the ability to induce directed migration. However, little is known about the role of cell‐substrate adhesion in mechanotransduction of D. discoideum. We hypothesized that adhesion is essential for the ability of cells to sense and respond to mechanical cues. To test this hypothesis, we examined whether mechanotransduction depends on SibA, a transmembrane protein involved in the adhesion of D. discoideum. Before testing cell response to shear flow, we studied the adhesion of SibA‐null cells grown under our experimental conditions. Adhesion of SibA‐null cells in growth media was significantly reduced compared to wild‐type cells, consistent with previously published findings. Notably, adhesion was significantly decreased by 25% in SibA‐null compared to wild‐type cells grown on a bacterial lawn, which is the growth method used prior to the assessment of response to shear flow. To test how SibA‐null cells respond to acute mechanical stimulation, we examined the localization of active Ras as a read‐out of the activation of the signal transduction network involved in directed migration. Both wild‐type and SibA‐null cells had a comparable response to 2‐second stimulation with shear flow at two different pressures. These findings suggest that the modest reduction in adhesion observed in SibA‐null cells did not affect the ability of cells to respond to shear flow stimulation. Whether more substantial changes in adhesion would perturb mechanotransduction in this system remains to be examined.

Investigation of the Adhesion‐Modulating Properties of Bovine Serum Albumin (BSA) in Dictyostelium discoideum

Dictyostelium discoideum is a social amoeba commonly used as a model organism in cellular migration studies. Dictyostelium adhesion is mediated by nonspecific interactions, such as Van der Waals forces, as well as specific protein‐mediated interactions between the cell and the substrate. Since adhesive properties of this organism affect its motility, finding ways to manipulate its nonspecific or specific interactions can be useful for future migration experiments. We tested the effects of bovine serum albumin (BSA), which is known to affect nonspecific binding via its interference with electrostatic interactions, on Dictyostelium adhesion. Bacterially‐grown wild‐type cells had increased adhesion when plated on 3% BSA, while axenically‐grown cells exhibited a decrease in adhesion on BSA. To explore why BSA has differential effects on adhesion of Dictyostelium grown under different conditions we tested adhesion of cells lacking one of the specific mediators of attachment, Talin A (TalA). Unlike wild‐type cells, both axenically and bacterially‐grown TalA‐null cells had decreased adhesion on BSA. These data suggest that BSA effects on adhesion may be dependent on TalA in bacterially‐grown cells. Alternatively, since TalA‐null cells have an impaired ability to phagocytose, they may behave more like axenically‐grown cells even when grown on bacteria. In this case, differential effects of BSA may be due to the inherent differences in the adhesion mechanism of cells grown under axenic vs. bacterially‐fed conditions. We are currently examining these possibilities.

Role of the Dimerization Domain of Filamin in Dictyostelium discoideum Response to Shear Flow

Molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation, which is critical in homeostasis and many diseases, are not well understood. Dictyostelium discoideum cells exposed to a brief burst of shear flow show rapid and transient activation of multiple components of the signal transduction network that participates in directed migration of these cells. Previous data from our laboratory demonstrated that actin crosslinking protein filamin is involved in the ability of cells to respond to shear flow. We also found that the actin‐binding domain is required for filamin’s function in this context. To determine if the dimerization domain (DD) is also required for filamin’s role in sensing/transmitting mechanical stimuli we generated a truncation construct of filamin lacking DD (FLNΔDD) and expressed it in wild‐type or filamin‐null cells. We found that FLNΔDDwas able to relocalize to the cortex of both wild‐type and filamin‐null cells following 2‐sec stimulation with shear flow, suggesting that dimerization between filamin molecules is not required for their recruitment to the cortex. To detect activation of the signal transduction network in the presence or absence of FLNΔDD, we used fluorescently‐tagged Ras binding domain biosensor that detects active Ras and was previously shown to relocalize to the cortex following mechanical stimulation. Surprisingly, FLNΔDDwas able to rescue the reduced response of filamin‐null cells to shear flow stimulation, suggesting that dimerization of filamin may not be needed for its ability to sense or transmit mechanical cues. However, the presence of FLNΔDD may also alter the overall organization of the cytoskeletal network, potentially explaining changes in mechanotransduction. We are currently investigating these possibilities.