Actin Filament Density Research Articles

In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography

Actin mediates insulin secretion in pancreatic β-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E β-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a “netlike” to a “blooming” architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.

Yes-associated protein regulates cortical actin architecture and dynamics through intracellular translocation of Rho GTPase-activating protein 18.

Yes-associated protein (YAP) is a transcriptional co-activator that controls the transcription of target genes and modulates the structures of various cytoskeletal architecture as mechanical responses. Although it has been known that YAP regulates actin-regulatory proteins, the detailed molecular mechanism of how they control and coordinate intracellular actin architecture remains elusive. Herein, we aimed to examine the structure and dynamics of intracellular actin architecture from molecular to cellular scales in normal and YAP-knockout (YAP-KO) cells utilizing high-speed atomic force microscopy (HS-AFM) for live-cell imaging and other microscope-based mechanical manipulation and measurement techniques. YAP-KO Madin-Darby canine kidney cells had a higher density and turnover of actin filaments in the cell cortex and a higher elastic modulus. Laser aberration assay demonstrated that YAP-KO cells were more resistant to damage than normal cells. We also found that Rho GTPase-activating protein 18 (ARHGAP18), a downstream factor of YAP, translocated from the cortex to the edge of sparsely cultured YAP-KO cells. It resulted in high RhoA activity and promotion of actin polymerization in the cell cortex and their reductions at the edge. HS-AFM imaging of live cell edge and a cell-migration assay demonstrated lower membrane dynamics and motility of YAP-KO cells than those of normal cells, suggesting lower actin dynamics at the edge. Together, these results demonstrate that a YAP-dependent pathway changes the intracellular distribution of RhoGAP and modulates actin dynamics in different parts of the cell, providing a mechanistic insight into how a mechano-sensitive transcription cofactor regulates multiple intracellular actin architecture and coordinates mechano-responses.

Increased endothelial sodium channel activity by extracellular vesicles in human aortic endothelial cells: putative role of MLP1 and bioactive lipids

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.

Arabidopsis ADF1 is Regulated by MYB73 and is Involved in Response to Salt Stress Affecting Actin Filament Organization.

Actin cytoskeleton and transcription factors play key roles in plant response to salt stress; however, little is known about the link between the two regulators in response to salt stress. Actin-depolymerizing factors (ADFs) are conserved actin-binding proteins in eukaryotes. Here, we revealed that the expression level of ADF1 was induced by salt stress. The adf1 mutants showed significantly reduced survival rate, increased percentage of actin cable and reduced density of actin filaments, while ADF1 overexpression seedlings displayed the opposite results when compared with WT under the same condition. Furthermore, biochemical assays revealed that MYB73, a R2R3 MYB transcription factor, binds to the promoter of ADF1 and represses its expression via the MYB-binding site core motif ACCTAC. Taken together, our results indicate that ADF1 participates in salt stress by regulating actin organization and may also serve as a potential downstream target of MYB73, which is a negative regulator of salt stress.

Numerical analysis of the impact of cytoskeletal actin filament density alterations onto the diffusive vesicle-mediated cell transport.

The interior of a eukaryotic cell is a highly complex composite material which consists of water, structural scaffoldings, organelles, and various biomolecular solutes. All these components serve as obstacles that impede the motion of vesicles. Hence, it is hypothesized that any alteration of the cytoskeletal network may directly impact or even disrupt the vesicle transport. A disruption of the vesicle-mediated cell transport is thought to contribute to several severe diseases and disorders, such as diabetes, Parkinson's and Alzheimer's disease, emphasizing the clinical relevance. To address the outlined objective, a multiscale finite element model of the diffusive vesicle transport is proposed on the basis of the concept of homogenization, owed to the complexity of the cytoskeletal network. In order to study the microscopic effects of specific nanoscopic actin filament network alterations onto the vesicle transport, a parametrized three-dimensional geometrical model of the actin filament network was generated on the basis of experimentally observed filament densities and network geometries in an adenocarcinomic human alveolar basal epithelial cell. Numerical analyzes of the obtained effective diffusion properties within two-dimensional sampling domains of the whole cell model revealed that the computed homogenized diffusion coefficients can be predicted statistically accurate by a simple two-parameter power law as soon as the inaccessible area fraction, due to the obstacle geometries and the finite size of the vesicles, is known. This relationship, in turn, leads to a massive reduction in computation time and allows to study the impact of a variety of different cytoskeletal alterations onto the vesicle transport. Hence, the numerical simulations predicted a 35% increase in transport time due to a uniformly distributed four-fold increase of the total filament amount. On the other hand, a hypothetically reduced expression of filament cross-linking proteins led to sparser filament networks and, thus, a speed up of the vesicle transport.

Deciphering the dynamics of lamellipodium in a fish keratocytes model

Motile cells depend on an intricate network of feedback loops that are essential in driving cell movement. Integrin-based focal adhesions (FAs) along with actin are the two key factors that mediate such motile behaviour. Together, they generate excitable dynamics that are essential for forming protrusions at the leading edge of the cell and, in certain cases, traveling waves along the membrane. A partial differential equation (PDE) model of a self-organizing lamellipodium in crawling keratocytes has been previously developed to understand how the three spatiotemporal patterns of activity observed in such cells, namely, stalling, waving and smooth motility, are produced. The model consisted of three key variables: the density of barbed actin filaments, newly formed FAs called nascent adhesions (NAs) and VASP, an anti-capping protein that gets sequestered by NAs during maturation. Using parameter sweeping techniques, the distinct regimes of behaviour associated with the three activity patterns were identified. In this study, we convert the PDE model into an ordinary differential equation (ODE) model to examine its excitability properties and determine all the patterns of activity exhibited by this system. Our results reveal that there are two additional regimes not previously identified, including bistability and oscillatory-like type IV excitability (generated by three steady states and their manifolds, rather than limit cycles). These regimes are also present in the PDE model. Applying slow-fast analysis on the ODE model shows that it exhibits a canard explosion through a folded-saddle and that rough motility seen in keratocytes is likely due to noise-dependent motility governed by dynamics near the interface of bistability and type IV excitability. The two parameter bifurcation suggests that the increase in the proportion of rough motion is due to a shift in activity towards the bistable and type IV excitable regimes induced by a decrease in NA maturation rate. Our results thus provide important insight into how microscopic mechanical effects are integrated to produce the observed modes of motility.

In Silico Model of Myosin VA-Mediated Liposome Transport Predicts Actin Filament Density and Liposome Diameter Dictate Transport Modes

In cells, myosin Va (myoVa) motor teams transport membrane-bound cargo through three-dimensional (3D) actin filament networks; a process we modeled both in vitro and in silico by characterizing myoVa liposome transport within 3D actin networks (Lombardo et al. 2017, 2019). Here, we developed a course-grained agent-based model to predict how actin filament density and liposome diameter impact the modes of myoVa-based transport of 5,000 fluid-like liposomes through a randomly oriented actin filament network. The modes of transport were characterized as diffusive, directed, or stationary. Each simulated liposome trajectory started at the center of the actin network with the myoVa-liposome not engaged to actin, and ended after 100 seconds unless the liposome moved 10 microns within this time. As a function of actin filament density, the liposome transport modes were mostly: 1) diffusive at low density; 2) directed along actin filaments at intermediate density; 3) stationary or effectively tethered at high density as motors on the liposome surface engage with multiple actin filaments in an effective tug of war. Transition from the directed to the tethered mode of transport with increasing actin filament density arises from a percolation phase transition in the actin network, which occurs at a critical actin filament density. Below this critical density, liposomes primarily interact with single actin filaments and are thus transported in a directed manner. Above this critical density, liposomes can engage multiple actin filaments simultaneously, resulting in liposomes becoming tethered. Increasing liposome diameter decreases the critical actin filament density at which this percolation phase transition occurs. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

Regulation of axon growth by myosin II-dependent mechanocatalysis of cofilin activity.

Serotonin (5-HT) is known to increase the rate of growth cone advance via cofilin-dependent increases in retrograde actin network flow and nonmuscle myosin II activity. We report that myosin II activity is regulated by PKC during 5-HT responses and that PKC activity is necessary for increases in traction force normally associated with these growth responses. 5-HT simultaneously induces cofilin-dependent decreases in actin network density and PKC-dependent increases in point contact density. These reciprocal effects facilitate increases in traction force production in domains exhibiting decreased actin network density. Interestingly, when PKC activity was up-regulated, 5-HT treatments resulted in myosin II hyperactivation accompanied by catastrophic cofilin-dependent decreases in actin filament density, sudden decreases in traction force, and neurite retraction. These results reveal a synergistic relationship between cofilin and myosin II that is spatiotemporally regulated in the growth cone via mechanocatalytic effects to modulate neurite growth.

Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity

The cell's dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

The Anticancer Activity of Cetraria Islandica (L.) Ach in Breast Cancer Cells Through Crosstalk of Ampk-α1 and Erk1/2 Signalling

In the present study, we aimed to evaluate the anticancer activities of Cetraria islandica (C.islandica) extracts on MCF-7 breast cancer cell lines. Cell viability, protein levels, apoptotic cells number, F-actin distribution were measured. Cell viability of MCF-7 breast cancer cells was found to be reduced in a dose-dependent manner.EC50 values of C.islandica on MCF-7 cells were found to be 9.2047 E-5 g/ml (cell amount) by using intelligence system. Expressions of p53, caspase 3 and Bcl-2, were shown to be elevated after low doses of extract and diminished after high dose treatments. PPAR- protein level was decreased, although AMP-activated kinases-α1 (AMPK-α1) protein level was increasedin its extract groups. ERK1/2 protein level was also elevated in its extract groups. 125 mg/ml of extract treated cells show a low decrease in actin filament density. MCF-7 cells with C.islandica treatment for 24 h increased the apoptotic cell percentage, though the cells-treated with C.islandica for 48 was high necrotic cells percentage. Consequently, the C.islandica extract treatment causes to elevate ERK1/2 and AMPK-α1 protein levels, resulting in PPAR- and then triggers the apoptosis by modulation caspase-3 and P53 protein levels. Therefore, C.islandica might be a good candidate for anticancer tissue, especially soft tissue tumours.

Stem Cell Cytoskeletal Responses to Pulsatile Flow in Heart Valve Tissue Engineering Studies.

Heart valve replacement options remain exceedingly limited for pediatric patients because they cannot accommodate somatic growth. To overcome this shortcoming, heart valve tissue engineering using human bone marrow stem cells (HBMSCs) has been considered a potential solution to the treatment of critical congenital valvular defects. The mechanical environments during in vitro culture are key regulators of progenitor cell fate. Here, we report on alterations in HBMSCs, specifically in their actin cytoskeleton and their nucleus under fluid-induced shear stresses of relevance to heart valves. HBMSCs were seeded in microfluidic channels and were exposed to the following conditions: pulsatile shear stress (PSS), steady shear stress (SS), and no flow controls (n = 4/group). Changes to the actin filament structure were monitored and subsequent gene expression was evaluated. A significant increase (p < 0.05) in the number of actin filaments, filament density and angle (between 30° and 84°), and conversely a significant decrease (p < 0.05) in the length of the filaments were observed when the HBMSCs were exposed to PSS for 48 h compared to SS and no flow conditions. No significant differences in nuclear shape were observed among the groups (p > 0.05). Of particular relevance to valvulogenesis, klf2a, a critical gene in valve development, was significantly expressed only by the PSS group (p < 0.05). We conclude that HBMSCs respond to PSS by alterations to their actin filament structure that are distinct from SS and no flow conditions. These changes coupled with the subsequent gene expression findings suggest that at the cellular level, the immediate effect of PSS is to initiate a unique set of quantifiable cytoskeletal events (increased actin filament number, density and angle, but decrease in filament length) in stem cells, which could be useful in the fine-tuning of in vitro protocols in heart valve tissue engineering.

Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization

S-equol is one of gut bacterial metabolites produced from soybean isoflavone daizein. While S-equol is known to promote glucose-induced insulin secretion from pancreatic β cells, whether S-equol affects glucagon-like peptide-1 (GLP-1) secretion from enteroendoceine L cells remains unclear. Here we assessed the effect of S-equol on GLP-1 secretion from mouse enteroendocrine L cell line GLUTag cells. GLUTag cells expressed GPR30 and estrogen receptors, which are putative S-equol receptors. Application of S-equol induced an increase in intracellular Ca2+ levels via GPR30. However, S-equol did not enhance GLP-1 exocytosis, and long-term treatment of S-equol suppressed GLP-1 secretion. Moreover, immunocytochemistry revealed that S-equol increased the density of cortical actin filaments via G12/13 signaling under GPR30. These data suggest that S-equol prevents GLP-1 secretion as a result of competing regulation between Ca2+ mobilization and actin reorganization.

Myosin Va Vesicular Transport is Modulated by Actin Filament Density, Orientation, and Polarity in an In Vitro 3D Actin Network

Myosin Va Vesicular Transport is Modulated by Actin Filament Density, Orientation, and Polarity in an In Vitro 3D Actin Network

Affine and non-affine deformations quantified in cytoskeletal networks through three-dimensional form-finding model

Actin filaments and cross-linkers are main components of cytoskeletal networks in eukaryotic cells, and they support bending moments and axial forces respectively. A three-dimensional form-finding model is proposed in this work to investigate affine and non-affine deformations in cytoskeletal networks. In recent studies, modeling of cytoskeletal networks turns out to be a key piece in the cell mechanics puzzle. We used form-finding analysis to compute and analyze cytoskeletal models. A three-dimensional model is much more flexible and contains more elements than a two-dimensional model, and non-linear finite element analysis is difficult to converge. Thus, vector form intrinsic finite element analysis is employed here for valid results. The three-dimensional model reveals new behaviors beyond earlier two-dimensional models and better aligns with available data. Relative density of actin filaments and height of the form-finding model both play important roles in determining cytoskeletal stiffness, positively and negatively, respectively. Real cytoskeletal networks are quite mixed in terms of affine and non-affine deformations, which are quantified by internal strain energy in actin filaments and cross-linkers. Results are also influenced by actin filament relative density and height of the model. The three-dimensional form-finding model does provide much more room for intensive studies on cytoskeletal networks. In our future study, microtubules, fluidics, viscoelastic-plastic cross-linkers and even the whole cell model may be taken into account gradually to improve the cytoskeletal form-finding model.

FMNL formins boost lamellipodial force generation

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.

Myo1C and Endophilin Promote Kinesin-Driven Tubulation at Engineered Cytoskeletal Intersections

In cells, kinesin-driven membrane tubulation along microtubules (MT) typically initiates in regions with a locally high actin filament (AF) density, suggesting that membrane deformations may result from the cooperative activities of kinesin and myosin motors at cytoskeletal intersections. While kinesin-1 motors are likely the primary drivers of tubule extension, membrane-binding myosin-I motors have also been proposed to participate in tubulation and membrane deformation in vivo. We hypothesize that Myo1c anchors membrane vesicles at AF/MT intersections, providing a resistive force that enables effective kinesin-1-driven tubulation. We tested this proposal by attaching kinesin-1 and Myo1c motors to giant unilamellar vesicles (GUVs); a truncated, His-tagged kinesin-1 construct (K560) was bound to NTA(Ni)-functionalized lipids and full length Myo1c was bound to PtdIns(4,5)P2 incorporated into the membrane. GUVs were introduced into chambers with engineered cytoskeletal arrays of sparse MTs bridging stripes densely-coated with short AFs. In the absence of Myo1c, kinesin-driven vesicles paused periodically at AF/MT intersections, but vesicle deformation was not observed. In contrast, GUVs with bound kinesin-1 and Myo1c motors paused at AF/MT intersections and deformed readily along MTs. However, extended tubules of diffraction-limited diameter were rarely observed. Next, we tested whether the addition of a BAR domain protein would facilitate vesicle tubulation by adding the membrane-binding domain of endophilin-A1, which is necessary for many endocytic trafficking tubulation events in vivo. We noted a significant, concentration-dependent, increase in the frequency and length of GUV tubulation events in the presence of endophilin. Importantly, this increased tubulation frequency required both kinesin-1 and Myo1c. These data provide insights into the interplay between cytoskeletal motors and BAR domain proteins in facilitating membrane tubulation. This work was supported by NIH grants GM087253 to E.M.O. and E.L.F.H. and T32-AR053461 to B.B.M.

Leukocytes Breach Endothelial Barriers by Insertion of Nuclear Lobes and Disassembly of Endothelial Actin Filaments

The endothelial cytoskeleton is a barrier for leukocyte transendothelial migration (TEM). Mononuclear and polymorphonuclear leukocytes generate gaps of similar micron-scale size when squeezing through inflamed endothelial barriers invitro and invivo. To elucidate how leukocytes squeeze through these barriers, we co-tracked the endothelial actin filaments and leukocyte nuclei in real time. Nuclear squeezing involved either preexistent or de novo-generated lobes inserted into the leukocyte lamellipodia. Leukocyte nuclei reversibly bent the endothelial actin stress fibers. Surprisingly, formation of both paracellular gaps and transcellular pores by squeezing leukocytes did not require Rho kinase or myosin II-mediated endothelial contractility. Electron-microscopic analysis suggested that nuclear squeezing displaced without condensing the endothelial actin filaments. Blocking endothelial actin turnover abolished leukocyte nuclear squeezing, whereas increasing actin filament density did not.We propose that leukocyte nuclei must disassemble the thin endothelial actin filaments interlacedbetween endothelial stress fibers in order to complete TEM.

Expression of MAX2 under SCARECROW promoter enhances the strigolactone/MAX2 dependent response of Arabidopsis roots to low-phosphate conditions.

MAX2/strigolactone signaling in the endodermis and/or quiescent center of the root is partially sufficient to exert changes in F-actin density and cellular trafficking in the root epidermis, and alter gene expression during plant response to low Pi conditions. Strigolactones (SLs) are a new group of plant hormones that regulate different developmental processes in the plant via MAX2, an F-box protein that interacts with their receptor. SLs and MAX2 are necessary for the marked increase in root-hair (RH) density in seedlings under conditions of phosphate (Pi) deprivation. This marked elevation was associated with an active reduction in actin-filament density and endosomal movement in root epidermal cells. Also, expression of MAX2 under the SCARECROW (SCR) promoter was sufficient to confer SL sensitivity in roots, suggesting that SL signaling pathways act through a root-specific, yet non-cell-autonomous regulatory mode of action. Here we show evidence for a non-cell autonomous signaling of SL/MAX2, originating from the root endodermis, and necessary for seedling response to conditions of Pi deprivation. SCR-derived expression of MAX2 in max2-1 mutant background promoted the root low Pi response, whereas supplementation of the synthetic SL GR24 to these SCR:MAX2 expressing lines further enhanced this response. Moreover, the SCR:MAX2 expression led to changes in actin density and endosome movement in epidermal cells and in TIR1 and PHO2 gene expression. These results demonstrate that MAX2 signaling in the endodermis and/or quiescent center is partially sufficient to exert changes in F-actin density and cellular trafficking in the epidermis, and alter gene expression under low Pi conditions.

Local Forces in Actin Networks with Gradients

Over the last two decades, experimental results have shown the response of uniform entangled actin networks to local stresses and strains is nonlinear. One theory proposes that nonlinear stresses associated with large network strains arise from gradients in polymer density in the network that can exert a net entropic forces on micron sized beads embedded in the network. In order to better understand the physics of actin polymer gradients, we probe the theory in reverse. We use a microfluidics device and photo-uncaging of salt and ATP to generate a gradient in actin filament density. We track the motion of neutrally buoyant beads embedded in the network. We do not observe a bias in the beads motion down the gradient as suggested by the entropic force model, however we do measure an overall enhancement of the bead mobility in the gradient compared with isotropic actin networks at the same concentration. We compare these results with a dissipative particle dynamics implementation of the experiment and polymer theory.

Abstract 1409: Whole-cell and local viscoelasticity of stem-like and non-stem-like breast cancer cells

Abstract It has been shown that the mechanical properties of pathological cells are different from healthy ones. Therefore, mechanical characterization of cells may be useful for investigating mechanisms and progression of diseases. In the case of cancer, the finding that cancerous cells are considerably more deformable than normal ones provides the motivation for investigating rheological differences between stem-like and non-stem-like cancer cells. In this study, the viscoelastic properties of six breast cancer cell lines, three of which have been regarded (based on surface markers) as stem-like and three considered less stem-like, were measured using micropipette aspiration and particle tracking microrheology. In micropipette experiments, cancer cells were aspirated one-by-one at controlled pressures into small glass micropipettes and the length of the aspirated section of the cells was tracked as a function of time. The applied pressure was either increased linearly or increased suddenly to different levels and then held constant. The homogeneous elastic half-space model was used to interpret the viscoelastic properties of the different cell lines. The effect of pressure loading rate on cell viscoelasticity was examined by applying three different aspiration pressure rates of 9, 6, and 3 Pa/s. The apparent Young's modulus was found to depend on pressure loading rate, which suggests a more complex rheology than a standard viscoelastic solid. Above a threshold force level, cells were also found to enter the micropipette continuously in a fashion that is analogous to white blood cells, i.e. as a liquid drop with constant cortical tension. A comparison between the Young's moduli showed that at least some of the stem-like breast cancer cell lines were less stiff than less stem-like cell lines and more likely to exhibit the liquid drop-like behavior. The reduction in stiffness and change in rheological regime might be the result of lower actin filament density which allowed the stem-like cancer cells to be more deformable while undergoing extravasation and intravasation during the metastatic process. Regional rheological differences were also found using particle tracking microrheology. Our hypothesis is that the differences in the rheological properties might help to distinguish between stem-like cancer cells and non-stem-like ones, which could eventually lead to a new detection and diagnostic method in cancer research. Citation Format: Amina Alipour, Monica M. Burdick, David F.J. Tees. Whole-cell and local viscoelasticity of stem-like and non-stem-like breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2015-1409