Dynacortin contributes to cortical viscoelasticity and helps define the shape changes of cytokinesis
2004; Springer Nature; Volume: 23; Issue: 7 Linguagem: Inglês
10.1038/sj.emboj.7600167
ISSN1460-2075
AutoresKristine D. Girard, Charles A. Chaney, Michael Delannoy, Scot C. Kuo, Douglas N. Robinson,
Tópico(s)Biocrusts and Microbial Ecology
ResumoArticle11 March 2004free access Dynacortin contributes to cortical viscoelasticity and helps define the shape changes of cytokinesis Kristine D Girard Kristine D Girard Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Charles Chaney Charles Chaney Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Michael Delannoy Michael Delannoy Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Scot C Kuo Scot C Kuo Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Douglas N Robinson Corresponding Author Douglas N Robinson Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Kristine D Girard Kristine D Girard Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Charles Chaney Charles Chaney Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Michael Delannoy Michael Delannoy Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Scot C Kuo Scot C Kuo Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Douglas N Robinson Corresponding Author Douglas N Robinson Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Author Information Kristine D Girard1, Charles Chaney2, Michael Delannoy1, Scot C Kuo2 and Douglas N Robinson 1 1Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA *Corresponding author. Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2196, USA. Tel.: +410 502 2850; E-mail: [email protected] The EMBO Journal (2004)23:1536-1546https://doi.org/10.1038/sj.emboj.7600167 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis. Introduction Cytokinesis is the mechanical process that cleaves a mother cell into two daughter cells and is essential for the propagation of cells across all phylogeny (Robinson and Spudich, 2000b). After more than 100 years of study, the fundamental mechanical bases for metazoan cytokinesis are still not understood, let alone the molecular bases for these mechanical elements (Rappaport, 1996). Two predominant models have been debated in recent decades: equatorial stimulation and polar relaxation (White and Borisy, 1983; Devore et al, 1989). The equatorial stimulation model assigns active force production to the contractile ring, which is considered a myosin-II-driven process (Robinson et al, 2002b). In contrast, the polar relaxation model assigns the polar cortex as the major source of active force production. As the volume of the mother cell is conserved during division, expansion of the polar cortex leads to cleavage furrow ingression. The role of myosin-II in the polar relaxation model is to serve as a dynamic actin crosslinker, increasing the equatorial stiffness so that the cleavage furrow ingresses. Still, recent studies have been unable to clarify the mechanical mechanisms of cytokinesis. Myosin-II is required for cytokinesis in suspension culture, while adherent mitotic ameboid myosin-II mutant cells divide nearly normally (DeLozanne and Spudich, 1987; Knecht and Loomis, 1987). Pharmacological inhibition of the polar cortex with actin filament destabilizers inhibits furrow ingression, whereas introduction of the inhibitors in the furrow region accelerated ingression of the cleavage furrow (O'Connell et al, 2001). By atomic force microscopy, which measures the bending modulus perpendicular to the cell's surface, the mammalian cell equator was more stiff than the basal global stiffness of the cell, a property that is most likely ubiquitous among metazoan-like cytokineses (Matzke et al, 2001). These results suggest that the global and equatorial cortices have distinct and important roles in cytokinesis. Force-balance is a principle fundamental to all mechanical shape changes. We have proposed a force-balance hypothesis in which the amount of contractile force generated by the contractile ring is specified by the mechanical resistance provided by the global cortex. A significant component of our hypothesis is that different mechanical elements are generated by specific proteins and that genetic interactions between these proteins reveal how they modulate different mechanical elements (Robinson and Spudich, 2000a; Robinson, 2001; Robinson et al, 2002b). In the force-balance hypothesis, at least three idealized mechanical elements are involved in cell shape changes. These elements include an active equatorial constricting force, cortical stretch modulus, and cytoplasmic viscosity. The active constricting forces for cytokinesis are proposed to be produced largely by equatorially localized myosin-II (Robinson and Spudich, 2000b). The cortical stretch modulus is the energy cost for deforming the cell. The stretch modulus is generated by cortical actin filaments and crosslinkers, and has been shown to be a strong predictor of how much myosin-II is recruited to the cleavage furrow cortex (Robinson et al, 2002a). As actin crosslinkers are dynamic, the cell's cortex is neither purely viscous nor elastic but has both viscous and elastic characteristics (Sato et al, 1987; Wachsstock et al, 1993, 1994; Xu et al, 1998). Cytoplasmic viscosity is the energy cost for bulk flow and the ratio of the stretch modulus to the cytoplasmic viscosity is predicted to set the rate of furrow ingression (Zhang and Robinson, in preparation). Genetic studies using Dictyostelium discoideum have identified proteins that are predicted to influence cortical viscoelasticity during cytokinesis (Robinson and Spudich, 2000a; Weber et al, 2000). Efficient cytokinesis requires different actin filament crosslinking proteins with different cellular distributions (Robinson and Spudich, 2000a). Dynacortin is an actin filament bundling protein that was originally identified in a genetic selection for suppressors of mutants devoid of cortexillin-I, an actin crosslinking protein (Faix et al, 1996, 2001; Robinson and Spudich, 2000a). The suppression experiment was performed with a cDNA expression library and only the carboxyl-half (C181) of dynacortin suppressed cortexillin-I (Robinson and Spudich, 2000a). In contrast, overexpression of the full-length dynacortin not only failed to rescue cortexillin-I but it also induced a dominant cytokinesis defect (Robinson and Spudich, 2000a). Dynacortin is distributed around the cell cortex, while cortexillin-I is concentrated in the equatorial cortex (Weber et al, 1999; Robinson and Spudich, 2000a). The cellular distributions and the genetic interactions of cortexillin-I and dynacortin suggest that they cooperate with each other to modulate the cortical mechanics that drive the complex shape changes of cytokinesis. In this paper, we combine biochemistry, genetics, direct observation of cytokinesis, and cellular biophysics to dissect the function of dynacortin and the basis for its genetic interactions with cortexillin-I. A significant new technical development is our ability to quantify the mechanical impact that dynacortin and cortexillin-I have on cell cortices, using a new noninvasive technology, laser-tracking microrheology (LTM) (Gittes et al, 1997; Mason et al, 1997; McGrath et al, 2000; Yamada et al, 2000). Several observations emerge from this study. First, we report the first loss-of-function phenotype for dynacortin, assigning a role for dynacortin, like cortexillin-I, in modulating the cortical viscoelasticity of interphase cells and specifying the shapes of cytokinesis. Second, dynacortin has two independent actin crosslinking domains, amino-half (N173) and C181, that crosslink with different relative activities. Third, our data suggest that the activities of proteins that are predicted to have different kinetics of actin crosslinking may be distinguished in vivo using LTM. Finally and most significantly, this study provides essential groundwork for determining how global and equatorial actin crosslinking specifies the shapes and ultimately the dynamics of cytokinesis. Results Dynacortin and cortexillin-I have quantitative effects on cell growth To initiate this study, we characterized the growth effects of a dynacortin hairpin construct (dynhp) that silenced dynacortin expression. The expression of dynhp led to a complete loss of detectable dynacortin and resulted in mild changes in the rate of increase of cell number as compared to wild type and cortexillin-I controls (Figure 1C; Table 1). We examined the cell size and relative growth rates of cells expressing each of the dynacortin domains for direct comparison to dynhp (Figure 1B and C; Table I). By microscopy, full-length dynacortin induced a 'big-cell' phenotype in wild type (Figure 1B) and cortexillin-I (data not shown), as observed previously (Robinson and Spudich, 2000a). Interestingly, N173 also induced a similar phenotype. C181 cells were uniform in size and rescued the growth rate as shown previously (Figure 1B, Table I; Robinson and Spudich, 2000a). Figure 1.(A) The cartoon represents the dynacortin proteins studied. (B) Micrographs of wild-type (wt:pLD1) cells carrying the control plasmid pLD1A15SN or an expression plasmid for dynacortin hairpin (dynhp), full-length dynacortin (Dyn), N173, or C181; scale bar, 40 μm. (C) Western immunoblot using antidynacortin polyclonal antibodies of cells expressing various dynacortin constructs. pLD1 is the expression vector used to express each protein in Dictyostelium and control strains were transformed with the empty vector. Download figure Download PowerPoint Table 1. Quantification of cellular growth rates and cellular concentrations of dynacortin and its domains Strain Relative growth ratea (n) % Total protein (n) [monomer], μM [dimer]b, μM Wild type: pLD1A15SN [100%] (12) 0.03% (4) 2 1 cortI1151: pLD1A15SN 54% (12) 0.03% (4) 2 1 Wild type: dynhp 110% (6) 0.0% (8) — — cortI1151: dynhp 40% (6) 0.0% (8) — — Wild type: N173 96% (8) 0.4% (2) 50 NA cortI1151: N173 21% (6) 0.4% (4) 50 NA Wild type: C181 88% (14) 0.5% (4) 60 30 cortI1151: C181 80% (10) 0.5% (2) 60 30 Wild type: dynacortin 61% (8) 0.3% (3) 20 10 cortI1151: dynacortin 35% (8) 0.2% (3) 10 5 a Relative growth rates were measured as the increase in cell number in suspension culture. All strains were normalized to wild type: pLD1A15SN controls, which is bracketed to indicate that fact. b Dynacortin and C181 form dimers while N173 exists as monomers (Supplementary Figure 1; Supplementary Table 1). To compare directly the in vivo morphological, growth, and mechanical effects (below) with the affinities and activities measured in vitro, we quantified the expression levels of each dynacortin domain in wild-type and cortexillin-I mutant strains (see Materials and methods). The cellular concentrations are presented (Table I). Although some of the protein concentrations appear unusually high, in fact the cellular concentrations of each domain are consistent with the biochemical activities of each protein (below; compare Table I with Table II). Table 2. Summary of actin-binding parameters for dynacortin, N173, and C181 Protein KD1app, μM (n) KD2app, μM (n) (KD1app)2KD2app, μM3 Half-maximal concentrationsa Fluorescence assay Falling ball viscometry Dynacortin 8.7±1.3 (6) 1.3±0.2 (17) 98 0.5 μM 0.5–1 μM N173 9.4±1.3 (10) 6.1±0.9 (21) 540 60 μM 50–60 μM C181 19±2.9 (7) 2.0±0.4 (20) 720 8 μM 15 μM KD1app increased with increasing actin concentration. This is likely due to actin polymers getting buried in the actin bundle so that it was more difficult for dynacortins to saturate each available site. The listed KD1app data are based on 5-μM actin-binding isotherms. Data from first binding step fit a model of a single binding isotherm, and the same saturation stoichiometry was observed at every actin concentration. For the 5-μM actin data, the χ2 values obtained by comparing the data to a simulated square hyperbola using the measured KD1app value ranged from 0.05 to 0.1 (P>0.20). Thus, the model is not rejected and KD1 appears to be only [actin]-dependent, not [crosslinker]-dependent. KD2app is based on all data as this parameter did not appear to be [actin]-dependent over greater than an order of magnitude of actin concentrations. Both KD values are only apparent values as dynacortin proteins that are bound and forming crosslinks versus those that are only bound cannot be cleanly separated by cosedimentation analysis. a Concentrations are for dimeric dynacortin and C181 and monomeric N173. Dynacortin, N173, and C181 crosslink actin but with different activities To elucidate dynacortin's molecular mechanisms, we analyzed dynacortin, C181, and N173 for their hydrodynamic properties and interactions with actin filaments (Table II, Supplementary Results and Supplementary Figures 1–3). Hydrodynamically, dynacortin is a rod-shaped dimer and the core of dimerization resides in the C181 domain (Supplementary Figure 1, Supplementary Table 1). Both N173 and C181 bind and crosslink actin in vitro with similar apparent thermodynamics, but with different overall activities as determined by falling ball viscometry and quantitative fluorescence microscopy (Table II; Supplementary Figures 2, 3). Using actin cosedimentation, falling ball viscometry, and the fluorescence microscopy assay, we determined an activity order for actin crosslinking as: Dynacortin>C181>N173 (Table II). The half-maximal concentrations for bundle formation determined from the fluorescence assay and falling ball viscometry are in good agreement with the actual amounts of each protein expressed in cells (Compare Tables I and II). As N173 and C181 had similar thermodynamics but C181 appeared to be a more effective crosslinker than N173, we hypothesize that N173 may have faster on and off rates to achieve the same actin crosslinking equilibrium. Dynacortin and cortexillin-I have complementary distributions during cytokinesis; C181 and N173 are distributed between the cytoplasm and cortex We examined the subcellular localization of dynacortin, N173, and C181 in interphase and dividing cells. Each protein was detected in two ways: GFP-fusions (GFP-dynacortin, GFP-N173 or C181-GFP) were imaged in live interphase cells (Figure 2A), and untagged proteins were imaged by immunocytochemistry in fixed dividing cells (Figure 2B). Both types of detection gave similar results for the localization of each protein. Only GFP-dynacortin and overexpressed untagged dynacortin showed clear enrichment in the cortex (Figure 2A and B). Cells expressing dynacortin-hairpin (dynhp), which had >99% reduction in the dynacortin level, had only a low level of background fluorescence when examined by immunocytochemistry (Figure 2B). Endogenous dynacortin is distributed between the cytoplasm and cortex in dividing cells (Figure 2B). Overexpressed full-length dynacortin showed a clear cortical enrichment in dividing cells (Figure 2B). N173 showed some cortical localization by both methods (Figure 2A and B), while C181 appeared to be distributed evenly between the cortex and cytoplasm (Figure 2A and B). To compare the localization of dynacortin to cortexillin-I, we examined the distribution of a GFP-tagged cortexillin-I expressed in a cortexillin-I mutant cell line (cortI1151:GFP-cort; Figure 2C). Cortexillin-I localized to the contractile ring in agreement with previously published observations (Weber et al, 1999). Figure 2.Dynacortin, N173, and C181 are globally distributed during cytokinesis. (A) GFP-dynacortin, GFP-N173, and C181-GFP were examined in live wild-type interphase cells; scale bar, 10 μm. (B) Total dynacortin was imaged by immunocytochemistry of dividing wild-type cells carrying episomal expression plasmids using antidynacortin polyclonal antibodies. Endogenous dynacortin (wt:pLD1) and wild-type cells expressing dynacortin-hairpin (wt:dynhp), which reduced dynacortin expression by >99%, are also presented. As the N173 and C181 domains are overexpressed, the pattern that is revealed is largely due to the transgene product. (C) A cortexillin-I mutant cell complemented with a GFP-cortexillin-I construct (cortI1151:GFPcort) is shown to indicate the distribution of cortexillin-I during cytokinesis. Scale bar (10 μm) in B applies to B and C. Download figure Download PowerPoint Dynacortin and cortexillin-I control the morphology of cytokinesis To ascertain dynacortin's and cortexillin-I's role in cytokinesis, we examined the morphology of dividing cells (Figure 3). We monitored the velocity of furrow ingression, whether the cleavage furrow ingressed evenly from both sides and whether the daughter cells were equal in size (Table III). To monitor the kinetics of furrow ingression, we measured the time-dependent change of the furrow diameter and the length of the furrow, which ultimately formed a bridge before being severed (Figure 3A). Wild-type cells always formed a discrete bridge relatively early in the process, and the bridge length always started smaller than the furrow diameter (Figure 3B). As the furrow continued to constrict, at some point the cylindrical bridge length and diameter were equal to each other. The distance when the furrow length and diameter were equal is defined as the crossover distance (Dx). Subsequently, the bridge became longer than the diameter as the furrow diameter continued to decrease before being severed. Figure 3.Analysis of the morphology of cytokinesis of wt:pLD1, wt:dynhp, cortI1151:pLD1 and cortI1151:C181 cells. (A) Diagram depicts the furrow diameter (Df) and the furrow length (Lf), which were measured to determine the crossover distance, Dx. Dx is defined as the distance where Df equals Lf. (B) Wt:pLD1 cells form a distinct bridge relatively early during the process. (C) Wt:dynhp cells form a distinct bridge at a later stage and appear more rounded in general. (D) This cortI1151:pLD1 cell formed a bridge at a later stage and the furrow ingression was faster on one side than the other. (E) The morphology of the cortexillin-I mutant cytokinesis was rescued by dynacortin C181. The furrow ingressed symmetrically and a distinct bridge was formed at an earlier stage similar to wild type; scale bar, 10 μm. Download figure Download PowerPoint Table 3. Average velocity of furrow ingression and crossover distance for each strain Strain Velocitya (μm/s) (n) Cross-over length, Dxb (μm) (n) Symmetric daughtersc/symmetric furrowsd (n) Wild type: pLD1 0.029±0.0032 (21) 2.7±0.16 (21) 100%/100% (24) Wild type: dynhp 0.033±0.0023 (14) 1.5±0.20 (14) 88%/100% (17) cortI1151: pLD1 0.032±0.0030 (16) 2.2±0.19 (16) 80% (30)/50% (22) cortI1151: cortI 0.027±0.0027 (6) 2.5±0.068 (6) 100%/100% (6) cortI1151: C181 0.037±0.0026 (19) 3.0±0.14 (19) 100%/95% (19) a The velocity is the average velocity of furrow ingression. b Crossover length is an objective morpho-metric parameter in which the length of cleavage furrow bridge is equal to the diameter of the cleavage furrow. The crossover length serves to distinguish quantitatively morphologies between different strains. Values are mean±s.e.m. Wild type:pLD1, cortI1151: C181 and cortI1151:cortI are all statistically indistinguishable. Wild type: dynhp (P<0.00005) and cortI1151: pLD1 (P<0.05) are significantly less than the wild-type control. cortI1151: C181 is significantly greater than cortI1151: pLD1 (0.0005<P<0.005). c The cells were assessed qualitatively as to whether the daughter cells were symmetric in size. d The furrows were assessed for being symmetrically positioned and whether they ingressed similarly from both sides. Deletion of cortexillin-I or silencing of dynacortin altered the morphology of cytokinesis (Figure 3C and D; Table III). Silencing of dynacortin caused the cells to appear more rounded during division and led to a large decrease in the crossover distance (Figure 3C; Table III). Deletion of cortexillin-I reduced the efficiency of cytokinesis, leading to a higher rate of failures, unequal cleavage events, and asymmetric ingression of the furrow (Table III). We focused on successful divisions so that the morphology (crossover distance and symmetry of furrow ingression) and velocities of furrow ingression could be fully assessed. Therefore, our data likely under-represent the unequal cleavage rate and cytokinesis failure rate as compared to other studies of cortexillin-I mutants (Weber et al, 2000). The crossover distance and symmetry of the cleavage furrows of cortexillin-I mutants were rescued by cortexillin-I (Table III) and dynacortin C181 (Figure 3E; Table III). Thus, even though dynacortin C181 and cortexillin-I have different cellular distributions during cytokinesis (Figure 2), both proteins quantitatively rescue the morphology of dividing cortexillin-I mutant cells. Laser-tracking microrheology allows quantitative analysis of cortical mechanics To quantify the effects of dynacortin and cortexillin-I on cortical mechanics, we used laser-tracking microrheology (LTM) to measure cortical viscoelasticity of wild-type and mutant cell lines (Figure 4; Mason et al, 1997; McGrath et al, 2000; Yamada et al, 2000). With LTM, the viscoelasticity of materials may be measured across a frequency spectrum by monitoring the motions of a spherical bead immersed in the material. Typically, viscoelastic moduli are measured when all dimensions of the material are nearly infinite relative to the bead. As cell cortices are sheet-like and often much thinner than the particles, we chose to measure the frequency-dependent viscoelasticity, ∣μ*∣, which is a phenomenological spring constant with viscous damping (see Methods for equations and rationale). Figure 4.Laser-tracking microrheology (LTM) system for measuring cortical mechanics. (A) The LTM system utilizes a low-power laser that is focused by the objective on a 0.7 μm polystyrene bead resting on the surface of the cell. As thermal energy drives the motion of the bead, the laser beam is deflected. The deflections are relayed via a condenser and relay lens to a quadrant photodiode detector, which monitors bead position. (B) Upper panel: To measure the cortical mechanical properties, a negatively charged polystyrene bead (arrow) is allowed to settle onto the surface of the cell, attaching nonspecifically. By focusing on the top surface of the cell, the bead is visible. Lower panel: By focusing on a lower cross-section, pseudopods can be seen. An arrow marks the XY-position of the bead. Download figure Download PowerPoint To determine cortical viscoelasticity, the thermal motions of a bead on a cell were monitored (at 6 kHz) for eleven 1-s intervals and then the viscoelasticity was calculated as the average of these 11 sequential measurements. For windows of time greater than 1 s, the bead's motions were often obscured by the cell's motion. Therefore, we restricted our observations to timescales shorter than 1 s. For physiological relevance, we focused on the longer timescales from 500-ms timescale (2 rad/s) to 5-ms timescale (200 rad/s). To verify that LTM of beads placed on the cell's extracellular surface would report on the underlying cytoskeleton, we compared wild-type cells to wild-type cells treated with latrunculin B, a potent F-actin depolymerizing agent (Figure 5A and B). In all, 85% of the viscoelasticity of wild-type cells was lost upon treatment with latrunculin B. As latrunculin B treatment results in depolymerization of filamentous actin, the 15% of viscoelasticity that remains presumably is due to the lipid bilayer and underlying cytosol. Figure 5.Dynacortin and its domains alter cortical viscoelasticity. (A) Frequency spectra of wild-type cells (wt:pLD1) and wt:pLD1 treated with latrunculin B show that actin filaments are necessary for 85% of cortical viscoelasticity. (B) Histograms of wt:pLD1 versus wt:pLD1 treated with latrunculin B at 10 and 100 rad/s for statistical comparison. n values are shown on bars. P-values from Student's t-test are <0.0001 at both frequencies. (C) Frequency spectra of viscoelasticity of wild-type and cortexillin-I mutant cells expressing dynacortin full-length, N173, C181, and cortexillin-I proteins. All spectra are the average spectra for each strain. The numbers of cells measured for each genetic strain are shown on the histrogram in part D. All measurements were made on 'wild-type'-sized cells to prevent possible complications due to cell size differences. The wild-type control spectrum is shown in each panel for direct comparison. (D) Histograms of means (±s.e.m.) for the viscoelasticity at 10 and 100 rad/s. These values correspond to the frequency spectra in (A). n values for each strain are listed on the bars in the histograms. For significance, the P-values from pairwise Student's t-test are presented in Supplemental Table 2. Download figure Download PowerPoint LTM also permits measurement of the phase angle, a property that reflects the solid-like or liquid-like nature of a material. As the material becomes more solid-like, the phase angle tends towards 0 and as the material becomes more liquid-like, the phase angle tends towards 90°. Pure actin networks and Cos7 lamellae have a characteristic phase angle of 22° (at 10 rad/s; Yamada et al, 2000). We extracted the phase angle data for each strain to determine how liquid- or solid-like the respective strains are. The average phase angles for all Dictyostelium strains ranged between 15 and 22°, which is similar to the phase angle of pure actin. In fact, none of the strains showed a difference greater than 2° across the full frequency range. In contrast, latrunculin B generally increased the phase angle (to around 30°) particularly at higher frequencies, indicating that the cell cortex was partially liquefied by the depolymerization of the actin filaments. Therefore, even though the beads are on the extracellular surface of the plasma membrane, the bead reports on a mechanical environment that is largely dependent on the cortical actin cytoskeleton and that has material properties (phase angle) similar to pure actin. Loss of dynacortin or cortexillin-I reduces cortical viscoelasticity By LTM analysis, removal of dynacortin and/or cortexillin-I had quantitative effects on cortical viscoelasticity (Figures 5 and 6; Supplementary Table 2). Deletion of cortexillin-I reduced the cortical viscoelasticity by approximately 30% across the full frequency spectrum as compared to the wild-type strain (Figure 5C, panel 1; 5D). The distribution of viscoelastic values of dynacortin silenced cells was much more heterogeneous with many more high-end outliers than wild-type parental cells were; therefore, we present histograms of viscoelastic measurements at two frequencies for wild-type and cortexillin-I mutant cells with and without dynacortin (Figure 6). Silencing of dynacortin in wild-type cells resulted in a 50% reduction in the median cortical viscoelasticity (Figure 6, panels 1, 2 versus 3, 4). Interestingly, the cortexillin-I background (Figure 6, panels 5, 6 versus 7, 8) seemed to be more resistant to the loss of dynacortin than the wild-type cells were. For example, silencing of dynacortin in wild-type cells reduced viscoelasticity from 0.28 to 0.14 nN/μm (at 10 rad/s), while in the cortexillin-I background, the numbers remai
Referência(s)