Lack of chromokinesin Klp-19 creates a more rigid midzone and affects force transmission during anaphase in C. elegans.

Recent studies have highlighted the significance of the spindle midzone - the region positioned between chromosomes - in ensuring proper chromosome segregation. By combining advanced 3D electron tomography and cutting-edge light microscopy we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and the chromokinesin KLP-19 in C. elegans suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length and turn-over of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length in the spindle midzone, which also leads to increased microtubule - microtubule interaction, thus building up a more robust microtubule network. The spindle is globally stiffer and more stable, which has implications for the transmission of forces within the spindle affecting chromosome segregation dynamics. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone.

The coordination of spindle-positioning forces during the asymmetric division of the Caenorhabditis elegans zygote.

In Caenorhabditis elegans zygote, astral microtubules generate forces essential to position the mitotic spindle, by pushing against and pulling from the cortex. Measuring microtubule dynamics there, we revealed the presence of two populations, corresponding to pulling and pushing events. It offers a unique opportunity to study, under physiological conditions, the variations of both spindle-positioning forces along space and time. We propose a threefold control of pulling force, by polarity, spindle position and mitotic progression. We showed that the sole anteroposterior asymmetry in dynein on-rate, encoding pulling force imbalance, is sufficient to cause posterior spindle displacement. The positional regulation, reflecting the number of microtubule contacts in the posterior-most region, reinforces this imbalance only in late anaphase. Furthermore, we exhibited the first direct proof that dynein processivity increases along mitosis. It reflects the temporal control of pulling forces, which strengthens at anaphase onset following mitotic progression and independently from chromatid separation. In contrast, the pushing force remains constant and symmetric and contributes to maintaining the spindle at the cell centre during metaphase.

Microtubule Feedback and LET-99-Dependent Control of Pulling Forces Ensure Robust Spindle Position.

During asymmetric division of the Caenorhabditis elegans zygote, to properly distribute cell fate determinants, the mitotic spindle is asymmetrically localized by a combination of centering and cortical-pulling microtubule-mediated forces, the dynamics of the latter being regulated by mitotic progression. Here, we show a, to our knowledge, novel and additional regulation of these forces by spindle position itself. For that, we observed the onset of transverse spindle oscillations, which reflects the burst of anaphase pulling forces. After delaying anaphase onset, we found that the position at which the spindle starts to oscillate was unchanged compared to control embryos and uncorrelated to anaphase onset. In mapping the cortical microtubule dynamics, we measured a steep increase in microtubule contact density after the posterior centrosome reached the critical position of 70% of embryo length, strongly suggesting the presence of a positional switch for spindle oscillations. Expanding a previous model based on a force-generator temporal control, we implemented this positional switch and observed that the large increase in microtubule density accounted for the pulling force burst. Thus, we propose that the spindle position influences the cortical availability of microtubules on which the active force generators, controlled by cell cycle progression, can pull. Importantly, we found that this positional control relies on the polarity-dependent LET-99 cortical band, the boundary of which could be probed by microtubules. This dual positional and temporal control well accounted for our observation that the oscillation onset position resists changes in cellular geometry and moderate variations in the active force generator number. Finally, our model suggests that spindle position at mitosis end is more sensitive to the polarity factor LET-99, which restricts the region of active force generators to a posterior-most region, than to microtubule number or force generator number/activity. Overall, we show that robustness in spindle positioning originates in cell mechanics rather than biochemical networks.

Membrane properties of Enchytraeus albidus originating from contrasting environments: a comparative analysis.

Ectothermic animals adapted to different environmental temperatures are hypothesized to have biological membranes with different chemical and physical properties such that membrane properties are optimized for their particular thermal environments. To test this hypothesis we analyzed the composition of phospholipid fatty acids (PLFAs) in seven different populations of Enchytraeus albidus originating from different thermal environments. The seven populations differ markedly in origin (polar-temperate) and are also characterized by marked difference in cold tolerance. The dominant PLFAs of E. albidus were C20:5, C20:4 and C20:2 (53-61% of total PLFA) followed by C18:0, C20:1 and C22:2 (17-20% of total PLFA). As hypothesized the PLFA composition varied significantly between populations and molar percentage of several of the PLFAs (particularly C18:2) correlated with the lower lethal temperature (LT50) of the seven populations. Unsaturation ratio (UFA/SFA) and average PLFA chain length also correlated significantly with LT50, such that cold sensitive populations had a shorter chain length and a lower UFA/SFA compared to cold tolerant populations. Reconstituted membranes of the least and most cold tolerant populations were used to compare membranes' physical properties by fluorescence anisotropy and bending rigidity. Measurements of anisotropy did not show any overall difference between populations with different cold tolerance. This could be interpreted as if E. albidus populations have achieved a similar "optimal" fluidity of the membrane with a somewhat different PLFA composition. Our study suggests that membrane lipid composition could be important for the cold tolerance of E. albidus; however, these differences are not easily differentiated in the measurements of the membranes' physical properties. Other parameters such as accumulation of glucose for cryoprotection and energy supply may also be important components of enchytraeid freeze tolerance.

Lipophilic contaminants influence cold tolerance of invertebrates through changes in cell membrane fluidity.

Contaminants taken up by living organisms in the environment as a result of anthropogenic contamination can reduce the tolerance of natural stressors, e.g., low temperatures, but the physiological mechanisms behind these interactions of effects are poorly understood. The tolerance to low temperatures of organisms that cannot regulate their body temperature (ectotherms) depends on their ability to increase the fluidity of their cellular membranes at low temperatures. Our study shows that contaminants accumulating in lipids of organisms alter the physical state of their membranes simply by being present. Contaminants of varying chemical structures can alter the membrane fluidity in either direction and correspondingly modulate the cold tolerance of intact animals.

Buffers affect the bending rigidity of model lipid membranes.

In biophysical and biochemical studies of lipid bilayers the influence of the used buffer is often ignored or assumed to be negligible on membrane structure, elasticity, or physical properties. However, we here present experimental evidence, through bending rigidity measurements performed on giant vesicles, of a more complex behavior, where the buffering molecules may considerably affect the bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on the bending modulus is observed in the presence of both salt and buffer molecules, which serves as a warning to experimentalists in the data interpretation of their studies, since typical lipid bilayer studies contain buffer and ion molecules.

Linking membrane physical properties and low temperature tolerance in arthropods.

Maintenance of membrane fluidity is of crucial importance in ectotherms experiencing thermal changes. This maintenance has in ectotherms most often been indicated using indirect measures of biochemical changes of phospholipid membranes, which is then assumed to modulate the physico-chemical properties of the membrane. Here, we measure bending rigidity characterizing the membrane flexibility of re-constituted membrane vesicles to provide a more direct link between membrane physical characteristics and low temperature tolerance. Bending rigidity of lipid bilayers was measured in vitro using Giant Unilamellar Vesicles formed from phospholipid extracts of the springtail, Folsomia candida. The bending rigidity of these membranes decreased when exposed to 0.4 vol% ethanol (0.23 mM/L). Springtails exposed to ethanol for 24h significantly increased their cold shock tolerance. Thus, by chemically inducing decreased membrane rigidity, we have shown a direct link between the physico-chemical properties of the membranes and the capacity to tolerate low temperature in a chill-susceptible arthropod.

Analysis of the shape fluctuations of reconstituted membranes using GUVs made from lipid extracts of invertebrates.

Changes in the physical properties of the lipid matrix of cell membranes have repeatedly been proposed to underlie stresses associated with e.g. drought, cold and xenobiotics. Therefore, the ability to experimentally monitor such properties is central to the fundamental physiological understanding of adaptive changes. Here, we test the analysis of shape fluctuations in membranes composed of lipid extracts from two soil invertebrates, and show that theories and experimental approaches previously developed for simpler liposomes may be applied directly to reconstituted membrane lipids. Specifically, we show how the bending rigidity of giant unilamellar liposomes of lipid extracts can be determined precisely. We suggest that future measurements of this parameter could elucidate mechanisms of adaptive processes such as changes in lipid composition and accumulation of protective osmolytes.

Intrinsic reaction-cycle time scale of Na+,K+-ATPase manifests itself in the lipid-protein interactions of nonequilibrium membranes.

Interaction between integral membrane proteins and the lipid-bilayer component of biological membranes is expected to mutually influence the proteins and the membrane. We present quantitative evidence of a manifestation of the lipid-protein interactions in liposomal membranes, reconstituted with actively pumping Na(+),K(+)-ATPase, in terms of nonequilibrium shape fluctuations that contain a relaxation time, τ, which is robust and independent of the specific fluctuation modes of the membrane. In the case of pumping Na(+)-ions, analysis of the flicker-noise temporal correlation spectrum of the liposomes leads to τ ~/= 0.5 s, comparing favorably with an intrinsic reaction-cycle time of about 0.4 s from enzymology.

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers.

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of "impurities" into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.

Softening of POPC membranes by magainin.

Magainin 2 belongs to the family of peptides, which interacts with the lipid membranes. The present work deals with the effect of this peptide on the mechanical properties of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine Giant Unilamellar Vesicle, characterized by the bending stiffness modulus. The bending elastic modulus is measured by Vesicle Fluctuation Analysis at biologically relevant pH and physiological buffer conditions and shows a dramatic decrease with increasing peptide concentration. The observed bilayer softening is interpreted in terms of a continuum model describing perturbations on the membrane organization. Our analysis suggests that the adsorbed peptides give rise to considerable local curvature disruptions of the membrane.

Giant unilamellar vesicle formation under physiologically relevant conditions.

We present an upgrade to the giant unilamellar vesicle (GUV) electroformation method allowing easy GUV production in different buffers and with various membrane compositions. Our experimental results reveal that lipid deposits obtained from aqueous liposome or proteoliposome dispersions are highly efficient for GUV electroformation. This is related to the ability of such dispersions to produce readily well-oriented membrane stacks. Furthermore, we present a protocol for GUV electroformation in various aqueous media, including electrolyte-containing buffers at characteristic concentrations of biological fluids. This work unlocks historical barriers to GUV applications in scientific fields like biology, biochemistry, or biophysics where membrane composition, as well as its aqueous environment, should be adapted to biological significance.