Cytoplasmic Flow Research Articles

Simultaneous Regulation of Cytokinetic Furrow and Nucleus Positions by Cortical Tension Contributes to Proper DNA Segregation during Late Mitosis.

Coordinating mitotic spindle and cytokinetic furrow positioning is essential to ensure proper DNA segregation. Here, we present a novel mechanism, which corrects DNA segregation defects due to cytokinetic furrow mispositioning during the first division of C.elegans embryos. Correction of DNA segregation defects due to an abnormally anterior cytokinetic furrow relies on the concomitant and opposite displacements of the furrow and of the anterior nucleus toward the posterior and anterior poles of the embryo, respectively. It also coincides with cortical blebbing and an anteriorly directed cytoplasmic flow. Although microtubules contribute to nuclear displacement, relaxation of an excessive tension atthe anterior cortex plays a central role in the correction process and simultaneously regulates cytoplasmic flow as well as nuclear and furrow displacements. This work thus reveals the existence of a so-far uncharacterized correction mechanism, which is critical to correct DNA segregation defects due to cytokinetic furrow mispositioning.

Antimicrobial peptides conjugated with fatty acids on the side chain of D-amino acid promises antimicrobial potency against multidrug-resistant bacteria

With the alarming burden of antibiotic resistance, antimicrobial peptides (AMPs) seem to be novel antimicrobial alternatives for infection treatment due to their rapid broad-spectrum antimicrobial activity and low tendency for bacterial resistance. To obtain promising AMPs, a series of new peptides were designed and synthesized by conjugating various lengths of fatty acid chains onto the side chain of the position 4 or 7 D-amino acid of Ano-D4,7 (analogue of anoplin with D-amino acid substitutions at positions 4 and 7). The new peptides exhibited excellent antimicrobial activity against a range of bacteria, especially multidrug-resistant bacteria in contrast to conventional antibiotics. Moreover, the new peptides conjugated with fatty acid chains ranging from 8 to 12 carbons in length presented preferable antimicrobial selectivity and anti-biofilm activity. Additionally, the new peptides also exerted high stability to trypsin, serum, salts and different pH environments. Most notably, the new peptides showed a low tendency to develop bacterial resistance and they displayed optimal antimicrobial activity against the obtained resistant strains. Furthermore, the results from the outer/inner membrane permeabilization and cytoplasmic membrane depolarization assays and flow cytometry and scanning electron microscopy analyses demonstrated that the new peptides exert antimicrobial effects by typical non-receptor-mediated membrane mechanisms, as well as intracellular targets characterized by gel retardation and reactive oxygen species (ROS) generation assays. Furthermore, the new peptides presented remarkable in vivo antimicrobial potency, anti-inflammatory activity, and endotoxin neutralization. Collectively, the conjugation of fatty acids to the side chains of D-amino acids is a potential strategy for designing hopeful antimicrobial alternatives to tackle the risk of bacterial resistance.

Tail shape evolution dynamics of MDCK cells on fibronectin substrates

Cell migration plays an important role in many biological processes. Trailing main cell body motion, the cell tail can be formed and exhibits different shapes. In this work, we experimentally investigated the tail shape evolution dynamics of MDCK adherent epithelial cells on fibronectin substrates. We found that, tails can be formed, (1) actively by turning a protruding arm with lamellipodia into a tail after the cell body reverses its moving direction, or (2) passively starting from a small region at the tail edge anchored on the substrate to resist the forward motion of the cell. Depending on whether the tail is overstretched, a narrow tail similar to the overstretched giant vesicle, or a tent shape tail with a fat base, can be developed. Focal adhesion locations at the tail end determine the tail end width and shape symmetry. Tails mainly exhibit three stage retraction, with a higher speed middle stage sandwiched between two slower speed stages. The speed of the middle stage is determined by whether the focal adhesions at the tail end in the initial low speed gliding retraction stage can be released in time. The actin bundles formed on the tail edge affect tail width evolution. In the narrow tail fast retraction, the interplay of membrane-cortex and actin bundle tensions, actin filament polymerization/depolymerization, cytoplasmic pressure, retardation of cytoplasmic flow through the narrow tail neck, leads to the developments of observed tail shape instabilities: tail end bulging, pearling, and wiggling.

Enhanced blebbing as a marker for metastatic prostate cancer.

Highly metastatic prostate cancer cells flowing through a microfluidic channel form plasma membrane blebs: they form 27% more than normal cells and have a lower stiffness (about 50%). Hypo-osmotic stress assays (with osmolarity) show 22% more blebbing of highly metastatic than moderately metastatic and 30% more than normal cells. Plasma membrane blebbing is known to provide important metastatic capabilities to cancer cells by aiding cell detachment from the primary tumor site and increasing cell deformability to promote cell migration through the extracellular matrix. Increased blebbing was attributed by others to decreased phosphorylated ezrin, radixin, and moesin (ERM) (p-ERM) protein expression-p-ERMs bind the plasma membrane to the actin cortex and reduced p-ERM expression can weaken membrane-cortex attachment. Myosin II also influences blebbing as myosin's natural contraction generates tension in the actin cortex. This increases cellular hydrostatic pressure, causes cortex rupture, cytoplasm flow out of the cortex, and hence blebbing. Highly metastatic cells are surprisingly found to express similar ezrin and myosin II levels but higher moesin levels in comparison with lowly metastatic or normal cells-suggesting that their levels, contrary to the literature [G. Charras and E. Paluch, Nat. Rev. Mol. Cell Biol. 9(9), 730-736 (2008); J.-Y. Tinevez, U. Schulze, G. Salbreux, J. Roensch, J.-F. Joanny, and E. Paluch, Proc. Natl. Acad. Sci. U.S.A. 106(44), 18581-18586 (2009); M. Bergert, S. D. Chandradoss, R. A. Desai, and E. Paluch, Proc. Natl. Acad. Sci. U.S.A. 109(36), 14434-14439 (2012); E. K. Paluch and E. Raz: Curr. Opin. Cell Biol. 25(5), 582-590 (2013)], are not important in metastatic prostate cell blebbing. Our results show that reduced F-actin is primarily responsible for increased blebbing in these metastatic cells. Blebbing can thus serve as a simple prognostic marker for the highly incident and lethal metastatic prostate cancer.

Nuclear (Bio)physics in the Embryo

Deneke etal. (2019) discover that dynamic interactions of cell cycle and actomyosin contractility systems synchronize nuclear cleavages, generating a cytoplasmic flow that results in a spatially uniform distribution of zygotic nuclei in the early Drosophila embryo. This work underscores the importance of self-organizing mechanisms before the onset of zygotic transcription.

A lumped parameter model of endoplasm flow in Physarum polycephalum explains migration and polarization-induced asymmetry during the onset of locomotion.

The plasmodial slime mold Physarum polycephalum exhibits strong, periodic flow of cytoplasm through the veins of its network. In the special case of mesoplasmodia, a newly described starvation-induced, shape-constant morphotype, this periodic endoplasm streaming is the basis of locomotion. Furthermore, we presume that cytoplasm flow is also involved in signal transmission and signal processing. Mesoplasmodia motility resembles amoeboid locomotion. In contrast to other amoebae, however, mesoplasmodia move without extending pseudopods and retain a coherent, fan-shaped morphology throughout their steady locomotion. Attaining sizes of up to 2 mm2, mesoplasmodia are also much bigger than other amoebae. We characterize this particular type of locomotion and identify patterns of movement. By using the analogy between pulsatile fluid flow through a network of elastic tubes and electrical circuits, we build a lumped model that explains observed fluid flow patterns. Essentially, the mesoplasmodium acts as a low-pass filter, permitting only low-frequency oscillations to propagate from back to front. This frequency selection serves to optimize flow and reduces power dissipation. Furthermore, we introduce a distributed element into the lumped model to explain cell polarization during the onset of chemotaxis: Biochemical cues (internal or external) lead to a local softening of the actin cortex, which in turn causes an increased flow of cytoplasm into that area and, thus, a net forward movement. We conclude that the internal actin-enclosed vein network gives the slime mold a high measure of control over fluid transport, especially by softening or hardening, which in turn leads to polarization and net movement.

Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm.Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

Role of mechanical flow for actin network organization

The major cytoskeletal protein actin forms complex networks to provide structural support and perform vital functions in cells. In vitro studies have revealed that the structure of the higher-order actin network is determined primarily by the type of actin binding protein (ABP). By comparison, there are far fewer studies about the role of the mechanical environment for the organization of the actin network. In particular, the duration over which cells reorganize their shape in response to functional demands is relatively short compared to the in vitro protein polymerization time, suggesting that such changes can influence the actin network formation. We hypothesize that mechanical flows in the cytoplasm generated by exogenous and endogenous stimulation play a key role in the spatiotemporal regulation of the actin architecture. To mimic cytoplasmic streaming, we generated a circulating flow using surface acoustic wave in a microfluidic channel and investigated its effect on the formation of networks by actin and ABPs. We found that the mechanical flow affected the orientation and thickness of actin bundles, depending on the type and concentration of ABPs. Our computational model shows that the extent of alignment and thickness of actin bundle are determined by the balance between flow-induced drag forces and the tendency of ABPs to crosslink actin filaments at given angles. These results suggest that local intracellular flows can affect the assembly dynamics and morphology of the actin cytoskeleton. Statement of SignificanceSpatiotemporal regulation of actin cytoskeleton structure is essential in many cellular functions. It has been shown that mechanical cues including an applied force and geometric boundary can alter the structural characteristics of actin network. However, even though the cytoplasm accounts for a large portion of the cell volume, the effect of the cytoplasmic streaming flow produced during cell dynamics on actin network organization has not been reported. In this study, we demonstrated that the mechanical flow exerted during actin network organization play an important role in determining the orientation and dimension of actin bundle network. Our result will be beneficial in understanding the mechanism of the actin network reorganization occurred during physiological and pathological processes.

Fluid dynamics during bleb formation in migrating cells in vivo.

Blebs are cellular protrusions observed in migrating cells and in cells undergoing spreading, cytokinesis, and apoptosis. Here we investigate the flow of cytoplasm during bleb formation and the concurrent changes in cell volume using zebrafish primordial germ cells (PGCs) as an in vivo model. We show that bleb inflation occurs concomitantly with cytoplasmic inflow into it and that during this process the total cell volume does not change. We thus show that bleb formation in primordial germ cells results primarily from redistribution of material within the cell rather than being driven by flow of water from an external source.

Cationic Amphipathic Triazines with Potent Anti-bacterial, Anti-inflammatory and Anti-atopic Dermatitis Properties

The emergence of multi-drug resistant bacteria forces the therapeutic world into a position, where the development of new and alternative kind of antibiotics is highly important. Herein, we report the development of triazine-based amphiphilic small molecular antibacterial agents as mimics of lysine- and arginine-based cationic peptide antibiotics (CPAs). These compounds were screened against a panel of both Gram-positive and Gram-negative bacterial strains. Further, anti-inflammatory evaluation of these compounds led to the identification of four efficient compounds, DG-5, DG-6, DL-5, and DL-6. These compounds displayed significant potency against drug-resistant bacteria, including methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), and vancomycin-resistant E. faecium (VREF). Mechanistic studies, including cytoplasmic membrane depolarization, confocal imaging and flow cytometry suggest that DG-5, DG-6, and DL-5 kill bacteria by targeting bacterial membrane, while DL-6 follows intracellular targeting mechanism. We also demonstrate that these molecules have therapeutic potential by showing the efficiency of DG-5 in preventing the lung inflammation of lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. More interestingly, DL-6 exhibited impressive potency on atopic dermatitis (AD)-like skin lesions in BALB/c mice model by suppressing pro-inflammatory cytokines. Collectively, these results suggest that they can serve a new class of antimicrobial, anti-inflammatory and anti-atopic agents with promising therapeutic potential.

Fluid Flows Shaping Morphology

A dynamic self-organized morphology is the hallmark of network-shaped organisms like slime moulds and fungi. Organisms continuously re-organize their flexible, undifferentiated body plans to forage for food. Among these organisms the slime mould Physarum polycephalum has emerged as a model to investigate how organism can self-organize their extensive networks and act as a coordinated whole. Cytoplasmic fluid flows flowing through the tubular networks have been identified as key driver of morphological dynamics. Inquiring how fluid flows can shape living matter from small to large scales opens up many new avenues for research.

Lipid droplet motility and organelle contacts.

Lipid droplets (LDs) are fat storage organelles integral to energy homeostasis and a wide range of cellular processes. LDs physically and functionally interact with many partner organelles, including the ER, mitochondria, lysosomes, and peroxisomes. Recent findings suggest that the dynamics of LD inter-organelle contacts is in part controlled by LD intracellular motility. LDs can be transported directly by motor proteins along either actin filaments or microtubules, via Kinesin-1, Cytoplasmic Dynein, and type V Myosins. LDs can also be propelled indirectly, by hitchhiking on other organelles, cytoplasmic flows, and potentially actin polymerization. Although the anchors that attach motors to LDs remain elusive, other regulators of LD motility have been identified, ranging from modification of the tracks to motor co-factors to members of the perilipin family of LD proteins. Manipulating these regulatory pathways provides a tool to probe whether altered motility affects organelle contacts and has revealed that LD motility can promote interactions with numerous partners, with profound consequences for metabolism. LD motility can cause dramatic redistribution of LDs between a clustered and a dispersed state, resulting in altered organelle contacts and LD turnover. We propose that LD motility can thus promote switches in the metabolic state of a cell. Finally, LD motility is also important for LD allocation during cell division. In a number of animal embryos, uneven allocation results in a large difference in LD content in distinct daughter cells, suggesting cell-type specific LD needs.

Cytoplasmic flows in starfish oocytes are fully determined by cortical contractions.

Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis. We then mathematically solve the Stokes equation for flows inside a deforming sphere using the measured surface displacements as boundary conditions. Our theoretical predictions agree very well with the intracellular flows quantified by particle image velocimetry, proving that during this stage the starfish cytoplasm behaves as a simple Newtonian fluid on the micrometer scale. We calculate the pressure field inside the oocyte and find that its gradient is too small as to explain polar body extrusion, in contrast to earlier suggestions. Myosin II inhibition by blebbistatin confirms this conclusion, because it diminishes cell shape changes and hydrodynamic flow, but does not abolish polar body formation.

Comparative systematics and phylogeography of Quercus Section Cerris in western Eurasia: inferences from plastid and nuclear DNA variation.

Oaks (Quercus) comprise more than 400 species worldwide and centres of diversity for most sections lie in the Americas and East/Southeast Asia. The only exception is the Eurasian sect. Cerris that comprises about 15 species, most of which are confined to western Eurasia. This section has not been comprehensively studied using molecular tools. Here, we assess species diversity and provide a first comprehensive taxonomic and phylogeographic scheme of western Eurasian members of sect. Cerris using plastid (trnH-psbA) and nuclear (5S-IGS) DNA variation with a dense intra-specific and geographic sampling. Chloroplast haplotypes primarily reflected phylogeographic patterns originating from interspecific cytoplasmic gene flow within sect. Cerris and its sister section Ilex. We identified two widespread and ancestral haplotypes, and locally restricted derived variants. Signatures shared with Mediterranean species of sect. Ilex, but not with the East Asian Cerris oaks, suggest that the western Eurasian lineage came into contact with Ilex only after the first (early Oligocene) members of sect. Cerris in Northeast Asia had begun to radiate and move westwards. Nuclear 5S-IGS diversification patterns were more useful for establishing a molecular-taxonomic framework and to reveal hybridization and reticulation. Four main evolutionary lineages were identified. The first lineage is comprised of Q. libani, Q. trojana and Q. afares and appears to be closest to the root of sect. Cerris. These taxa are morphologically most similar to the East Asian species of Cerris, and to both Oligocene and Miocene fossils of East Asia and Miocene fossils of western Eurasia. The second lineage is mainly composed of the widespread Q. cerris and the narrow endemic species Q. castaneifolia, Q. look, and Q. euboica. The third lineage comprises three Near East species (Q. brantii, Q. ithaburensis and Q. macrolepis), well adapted to continental climates with cold winters. The forth lineage appears to be the most derived and comprises Q. suber and Q. crenata. Q. cerris and Q. trojana displayed high levels of variation; Q. macrolepis and Q. euboica, previously treated as subspecies of Q. ithaburensis and Q. trojana, likely deserve independent species status. A trend towards inter-specific crosses was detected in several taxa; however, we found no clear evidence of a hybrid origin of Q. afares and Q. crenata, as currently assumed.

Abscisic acid and nitric oxide modulate cytoskeleton organization, root hair growth and ectopic hair formation in Arabidopsis

Abscisic acid (ABA) and nitric oxide (NO) are two plant growth regulators that participate in many signaling cascades in different organs all along the plant life. Here, we were interested in deciphering the effects of ABA and NO on the cytoskeleton organization in a model of polarized cell growth like root hairs. Arabidopsis roots were exposed to different concentrations of ABA, and the length of primary root, epidermal cells and root hairs were measured. The NO concentration was detected with the NO-specific fluorescent probe DAF-FM DA. To quantify the effects of ABA and NO on cytoskeleton, Arabidopsis seedlings expressing GFP-MAP4 were used to analyze microtubules (MTs) orientation. Changes in cytoplasmic streaming were quantified through fluorescence recovery after photobleaching (FRAP) experiments using confocal laser scanning microscopy (CLSM) and the probe fluorescein diacetate (FDA). Results indicate that ABA decreases root hair length and induces the differentiation of atrichoblasts into trichoblasts, increasing root hair density. ABA also triggers an increase of NO level in root hairs. Both, ABA and NO affect MT organization in root hairs. While root hairs show MT orientation close to the longitudinal axis in control roots, ABA and NO treatments induce the oblique orientation of MTs. In parallel, cytoplasmic flow, executed by actin cytoskeleton, is enhanced by NO, in an ABA-independent manner. For all experimental conditions assayed, basal levels of NO are required to keep MT organization and cytoplasmic streaming. Our findings support ABA and NO as key modulators of growth and ectopic formation of root hairs through actions on cytoskeleton functions.

Microtubule-Dependent Confinement of a Cell Signaling and Actin Polymerization Control Module Regulates Polarized Cell Growth

Cell types with wildly varying shapes use many of the same signaling and cytoskeletal proteins to dynamically pattern their geometry [1-3]. Plant cells are encased in a tough outer cell wall, and growth patterns are indirectly controlled by the cytoskeleton and its ability to locally specify the material properties of the wall [4, 5]. Broad and non-overlapping domains of actin and microtubules are predicted to create sharp cell-wall boundaries with distinct mechanical properties [6] that are often proposed to direct growth patterns and cell shape [1, 6, 7]. However, mechanisms by which the cytoskeleton is patterned at the spatial and temporal scales that dictate cell morphology are not known. Here, we used combinations of live-cell imaging probes and unique morphology mutants in Arabidopsis to discover how the microtubule and actin systems are spatially coordinated to pattern polarized growth in leaf epidermal cells. The DOCK family guanine nucleotide exchange factor (GEF) SPIKE1 [8, 9] clusters and activates conserved heteromeric WAVE/SCAR and ARP2/3 complexes at the cell apex to generate organized actin networks that define general cytoplasmic flow patterns. Cortical microtubules corral punctate SPIKE1 signaling nodules and restrict actin polymerization within a broad microtubule-depletion zone at the cell apex. Our data provide a useful model for cell-shape control, in which a GEF, actin filament nucleation complexes, microtubules, and the cell wall function as interacting systems that dynamically pattern polarized growth.

Long-range interactions of Chara chloroplasts are sensitive to plasma-membrane H+ flows and comprise separate photo- and dark-operated pathways.

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H+ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H+ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1-2min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (QA) via two separate (photochemical and non-photochemical) pathways.

Fluid flows shaping organism morphology.

A dynamic self-organized morphology is the hallmark of network-shaped organisms like slime moulds and fungi. Organisms continuously reorganize their flexible, undifferentiated body plans to forage for food. Among these organisms the slime mould Physarum polycephalumhas emerged as a model to investigate how an organism can self-organize their extensive networks and act as a coordinated whole. Cytoplasmic fluid flows flowing through the tubular networks have been identified as the key driver of morphological dynamics. Inquiring how fluid flows can shape living matter from small to large scales opens up many new avenues for research. This article is part of the theme issue 'Self-organization in cell biology'.

Optical control of cytoplasmic flows.

Cytoplasmic flows are essential for various cellular processes. However, tools to manipulate these flows within cells are still lacking. Now research shows that an optical tool allows for control of cytoplasmic flows and can be used as a subcellular rheometer.

Non-invasive perturbations of intracellular flow reveal physical principles of cell organization.

Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS.