Cellular Cortex Research Articles

TGF-β2 enhances nanoscale cortex stiffness via condensation of cytoskeleton-focal adhesion plaque.

Physical spatiotemporal characteristics of cellular cortex dominate cell functions and even determine cell fate. The cellular cortex is able to re-organize to a dynamic steady status with changed stiffnesses once stimulated, and thus alter the physiological and pathological activities of almost all types of cells. TGF-β2, a potent pleiotropic growth factor, plays important roles in cartilage development, endochondral ossification, and cartilage diseases. However, it is not yet known whether TGF-β2 would alter the physical spatiotemporal characteristics of the cell cortex such as cortex stiffness, thereby affecting the function of chondrocytes. In this study, we investigated the influence of TGF-β2 on cellular cortex stiffness of chondrocytes and the underlying mechanism. We firstly detected TGF-β2-induced changes in cytoskeleton and focal adhesion plaque, which were closely related to cellular cortex stiffness. We then characterized the landscape of nanoscale cortex stiffness in individual chondrocytes induced by TGF-β2 via atomic force microscope (AFM). By using inhibitors, latrunculin A and blebbistatin, we verified the importance of cytoskeleton-focal adhesion plaque axis on cellular cortex stiffness of chondrocytes induced by TGF-β2. We finally elucidated that TGF-β2 enhanced the phosphorylation of Smad3 and facilitated the nuclear accumulation of p-Smad3. The p-Smad3 aggregated in the nuclei enhanced the cytoskeleton and focal adhesion plaque at transcriptional level, thereby mediating changes in cell cortex stiffness. Taken together, these results provide an understanding about the role of TGF-β2 on physical spatiotemporal properties of cell cortex in chondrocytes, and might provide cues for interpretation of cartilage development and interventions to cartilage diseases.

Polar targeting of proteins - a green perspective.

Cell polarity - the asymmetric distribution of molecules and cell structures within the cell - is a feature that almost all cells possess. Even though the cytoskeleton and other intracellular organelles can have a direction and guide protein distribution, the plasma membrane is, in many cases, essential for the asymmetric localization of proteins because it helps to concentrate proteins and restrict their localization. Indeed, many proteins that exhibit asymmetric or polarized localization are either embedded in the PM or located close to it in the cellular cortex. Such proteins, which we refer to here as 'polar proteins', use various mechanisms of membrane targeting, including vesicle trafficking, direct phospholipid binding, or membrane anchoring mediated by post-translational modifications or binding to other proteins. These mechanisms are often shared with non-polar proteins, yet the unique combinations of several mechanisms or protein-specific factors assure the asymmetric distribution of polar proteins. Although there is a relatively detailed understanding of polar protein membrane targeting mechanisms in animal and yeast models, knowledge in plants is more fragmented and focused on a limited number of known polar proteins in different contexts. In this Review, we combine the current knowledge of membrane targeting mechanisms and factors for known plant transmembrane and cortical proteins and compare these with the mechanisms elucidated in non-plant systems. We classify the known factors as general or polarity specific, and we highlight areas where more knowledge is needed to construct an understanding of general polar targeting mechanisms in plants or to resolve controversies.

Full-field optical coherence microscopy enables high-resolution label-free imaging of the dynamics of live mouse oocytes and early embryos

High quality label-free imaging of oocytes and early embryos is essential for accurate assessment of their developmental potential, a key element of assisted reproduction procedures. To achieve this goal, we propose full-field optical coherence microscopy (FF-OCM), constructed as a compact module fully integrated with a commercial wide-field fluorescence microscope. Our system achieves optical sectioning in wide-field, high in-plane resolution of 0.5 µm, and high sensitivity to backscattered light. To demonstrate its imaging capabilities, we study live mouse oocytes and embryos at all important stages of meiotic maturation and early embryogenesis. Our system enables visualization of intracellular structures, which are not visible in common bright-field microscopy, i.e., internal structure of nuclear apparatus, cytoskeletal filaments, cellular cortex, cytoplasmic protrusions, or zona pellucida features. Additionally, we visualize and quantify intracellular dynamics like cytoplasmic stirring motion, nuclear envelope fluctuations and nucleolus mobility. Altogether, we demonstrate that FF-OCM is a powerful tool for research in developmental biology that also holds great potential for non-invasive time-lapse monitoring of oocyte and embryo quality in assisted reproduction.

Ezrin, radixin, and moesin are dispensable for macrophage migration and cellular cortex mechanics

The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.

Unraveling the defensive strategies of camel thorn Alhagi maurorum medik. For thriving in arid and semi-arid environments

The study aimed to evaluate the role of morpho-physiological and anatomical attributes of Alhagi maurorum Medik. Populations from five water deficit regions in Punjab province, Pakistan, namely, Cholistan desert (KHP), Rajanpur (DGK), Thal (LYH), Patisar Lake (LAS), and Salt Range (WSM), in their adaptability to arid and semi-arid regions. The study sheds light on the adaptive components of various plant populations to cope with increasing dryness ratio. Arid populations [Khanpur-KHP (D = 44.91, P = 97.1 mm), Dera Ghazi Khan-DGK (D = 41.41, P = 105.3 mm), Layyah-LYH (D = 37.11, P = 117.5 mm) and Ladamsar-LAS (D = 34.83, P = 125.2 mm)] relied on larger roots and leaves, enhanced biomass production, ion accumulation, and photosynthetic pigments. It also encompassing larger root and stem cellular area, cortex and vascular region, thicker leaves, and numerous large stomata. Semi-arid populations display distinctive changes, such as longer shoots, higher root and shoot Na+ levels, organic osmolytes, thicker epidermis, and enlarged pith region in stems [Warcha Salt Mine-WSM (D = 8.70, P = 501 mm)]. These modifications at both structural and functional levels guarantee the survival and success of this species in challenging environmental conditionswhich will have implications for sustainable agriculture, ecosystem restoration, and adaptation to climate change adaptation in arid and semi-arid regions.

Periodicity, mixed-mode oscillations, and multiple timescales in a phosphoinositide-Rho GTPase network

While rhythmic contractile behavior is commonly observed at the cellular cortex, the primary focus has been on excitable or periodic events described by simple activator-delayed inhibitor mechanisms. We show that Rho GTPase activation in nocodazole-treated mitotic cells exhibits both simple oscillations and complex mixed-mode oscillations. Rho oscillations with a 20- to 30-s period are regulated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) via an activator-delayed inhibitor mechanism, while a slow reaction with period of minutes is regulated by phosphatidylinositol 4-kinase via an activator-substrate depletion mechanism. Conversion from simple to complex oscillations can be induced by modulating PIP3 metabolism or altering membrane contact site protein E-Syt1. PTEN depletion results in a period-doubling intermediate, which, like mixed-mode oscillations, is an intermediate state toward chaos. In sum, this system operates at the edge of chaos. Small changes in phosphoinositide metabolism can confer cells with the flexibility to rapidly enter ordered states with different periodicities.

Septin 9 Orients the Apico-Basal Polarity Axis and Controls Plasticity Signals.

The cytoskeleton is a master organizer of the cellular cortex and membrane trafficking and therefore plays a crucial role in apico-basal polarity. Septins form a family of GTPases that assemble into non-polar filaments, which bind to membranes and recruit cytoskeletal elements such as microtubules and actin using their polybasic (PB) domains, to perform their broad biological functions. Nevertheless, the role of septins and the significance of their membrane-binding ability in apico-basal polarity remains under-investigated. Here, using 3D cultures, we demonstrated that septin 9 localizes to the basolateral membrane (BM). Its depletion induces an inverted polarity phenotype, decreasing β-catenin at BM and increasing transforming growth factor (TGFβ) and Epithelial-Mesenchymal Transition (EMT) markers. Similar effects were observed after deleting its two PB domains. The mutant became cytoplasmic and apical. The cysts with an inverted polarity phenotype displayed an invasive phenotype, with src and cortactin accumulating at the peripheral membrane. The inhibition of TGFβ-receptor and RhoA rescued the polarized phenotype, although the cysts from overexpressed septin 9 overgrew and presented a filled lumen. Both phenotypes corresponded to tumor features. This suggests that septin 9 expression, along with its assembly through the two PB domains, is essential for establishing and maintaining apico-basal polarity against tumor development.

A computational model of self-organized shape dynamics of active surfaces in fluids

Mechanochemical processes on surfaces such as the cellular cortex or epithelial sheets, play a key role in determining patterns and shape changes of biological systems. To understand the complex interplay of hydrodynamics and material flows on such active surfaces requires novel numerical tools. Here, we present a finite-element method for an active deformable surface interacting with the surrounding fluids. The underlying model couples surface and bulk hydrodynamics to surface flow of a diffusible species which generates active contractile forces. The method is validated with previous results based on linear stability analysis and shows almost perfect agreement regarding predicted patterning. Away from the linear regime we find rich non-linear behavior, such as the presence of multiple stationary states. We study the formation of a contractile ring on the surface and the corresponding shape changes. Finally, we explore mechanochemical pattern formation on various surface geometries and find that patterning strongly adapts to local surface curvature. The developed method provides a basis to analyze a variety of systems that involve mechanochemical pattern formation on active surfaces interacting with surrounding fluids.

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion.

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides.

Partitioning of ribonucleoprotein complexes from the cellular actin cortex.

The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a fluorescence microscopy method with the capacity to infer the intracellular distribution of labeled biomolecules with subresolution accuracy. Unexpectedly, we find that RNA binding proteins are partially excluded from the cytoplasmic volume adjacent to the plasma membrane that corresponds to the actin cortex. Complementary diffusion measurements of RNA-protein complexes suggest that a rudimentary model based on excluded volume interactions can explain this partitioning effect. Our results suggest the actin cortex meshwork may play a role in regulating the biomolecular content of the volume immediately adjacent to the plasma membrane.

Cell-derived Giant Membrane Vesicles Enclosing the Anthracycline Anti-Cancer Drug Doxorubicin and their Cytotoxicity

Although giant membrane vesicles prepared from paraformaldehyde-treated mammalian cells have been used to elucidate lipid and protein dynamics on the cell surface, there are few studies that have used them as a tool to deliver drugs to cells. Here we found that anti-cancer drug doxorubicin (Dox) was efficiently incorporated into and stably retained in giant membrane vesicles prepared from HeLa human cervical cancer cells. Intriguingly, coincubation of Dox-enclosed giant membrane vesicles with human pancreatic cancer-derived PK-59 and gastric cancer-derived KE-39 cells led to cell death. Microscopic observation of vesicle-docked cells revealed that docking of at least one to a few Dox-enclosed giant vesicles on the cellular cortex was sufficient to induce cell death, suggesting applicability of cell-derived giant membrane vesicles as an efficient drug delivery vector in cultured cell systems.

The membrane-cortex crosslinker ezrin acts as a sliding anchor on F-actin

The cellular actin cortex is a thin meshwork of filamentous (F)-actin and nonmuscle myosin II that lies immediately beneath the cell membrane. The cortex exhibits fluid-like properties in that it can undergo rapid and dramatic changes in shape during cell protrusion generation and migration. At the same time, the cortex must remain stably linked to the cell membrane to protect the membrane from mechanical disruption. How these two seemingly opposed functions are achieved at the molecular level is unclear.

Basic structure and cytocompatibility of giant membrane vesicles derived from paraformaldehyde-exposed human cells.

Exposure of cultured mammalian cells to paraformaldehyde (PFA) is an effective approach to induce membrane blebs, which is followed by their detachment from the cellular cortex to yield giant membrane vesicles in extracellular spaces. Although PFA-induced giant vesicles have attracted significant interest in the field of cell membrane dynamics, their biochemical components and cytocompatibility remain largely unknown. In this report, we exposed human cervical cancer HeLa cells to PFA under metal-free buffer conditions to produce giant vesicles. We analyzed the components and structure of the purified PFA-induced giant vesicles. Co-culturing PFA-induced giant vesicles with exponentially growing HeLa cells resulted in docking of a significant number of the giant vesicles to the cell surface with seemingly no cytotoxicity. Intriguingly, we found that pre-treatment of HeLa cells with peptide-N-glycosidase and neuraminidase was effective in facilitating cellular uptake of constituents residing inside the vesicles. The results revealed further details about the effect of PFA on cell membranes and provide insights for studying the interaction between PFA-induced giant vesicles and cultured cells.

Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying.

The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.

Intra-bundle contractions enable extensile properties of active actin networks

The cellular cortex is a dynamic and contractile actomyosin network modulated by actin-binding proteins. We reconstituted a minimal cortex adhered to a model cell membrane mimicking two processes mediated by the motor protein myosin: contractility and high turnover of actin monomers. Myosin reorganized these networks by extensile intra‑bundle contractions leading to an altered growth mechanism. Hereby, stress within tethered bundles induced nicking of filaments followed by repair via incorporation of free monomers. This mechanism was able to break the symmetry of the previously disordered network resulting in the generation of extensile clusters, reminiscent of structures found within cells.

Cell Junction Mechanics beyond the Bounds of Adhesion and Tension.

Cell-cell junctions, in particular adherens junctions, are major determinants of tissue mechanics during morphogenesis and homeostasis. In attempts to link junctional mechanics to tissue mechanics, many have utilized explicitly or implicitly equilibrium approaches based on adhesion energy, surface energy, and contractility to determine the mechanical equilibrium at junctions. However, it is increasingly clear that they have significant limitations, such as that it remains challenging to link the dynamics of the molecular components to the resulting physical properties of the junction, to its remodeling ability, and to its adhesion strength. In this perspective, we discuss recent attempts to consider the aspect of energy dissipation at junctions to draw contact points with soft matter physics where energy loss plays a critical role in adhesion theories. We set the grounds for a theoretical framework of the junction mechanics that bridges the dynamics at the molecular scale to the mechanics at the tissue scale.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

Ovarian mucinous neoplasms, intestinal type, in premenopausal patients, develop in abnormal ovaries

Although several studies have addressed different aspects of mucinous neoplasms arising in the ovary, such as their clinicopathologic features, immunohistochemical profile, and molecular characteristics, no study has presented an analysis of the ovarian tissue where these neoplasms arise. In this study, we included 196 cases of intestinal-type ovarian mucinous neoplasms in premenopausal patients. Our main goal was to perform a rigorous examination of the ovarian tissue surrounding these neoplasms. We also reviewed the clinicopathologic features of these cases. For comparison, the background ovarian tissue in 85 cases of ovarian serous neoplasm and in 29 cases of metastatic neoplasms to the ovary, as well as 57 normal ovaries, was examined. All the patients in this study, which included those with mucinous and with serous neoplasms primary in the ovary, those with metastatic tumors to the ovaries, and those with normal ovaries, were also premenopausal. Patients affected by ovarian mucinous neoplasms ranged in age from 13 to 52 years (median = 36years). Nulligravidity was seen in 50%, 32%, and 22% of patients with mucinous carcinomas, mucinous borderline neoplasms, and mucinous cystadenomas, respectively. Ovarian mucinous intestinal neoplasms arise in abnormal ovaries characterized by two important features: (1) an abnormal ovarian cortex, seen in 95% of the cases, which is hypocellular or with no distinction between the cellular cortex and medulla, and (2) a remarkable paucity of primordial follicles. The abnormalities detected in the background ovarian tissue might provide insights into the tumorigenesis of these neoplasms and might facilitate their distinction from metastasis to the ovary, in premenopausal patients.

Fine structure of the central brain in the octopod Eledone cirrhosa (Lamarck, 1798) (Mollusca-Octopoda).

This study aims to investigate the fine structure of the different cell types in the central brain of Eledone cirrhosa; the organelles in the neurons and the glial cells; the glial hemolymph-brain barrier; the neuro-secretions and the relationships between glial and nerve cells. The brain is surrounded by a non-cellular neurilemma followed by a single layer of perilemmal cells. Ependymal cells, highly prismatic glial cells, astrocytes, oligodendrocytes and epithelial processes were observed. The perikarya of the neurons are filled with slightly oval nuclei with heterochromatin, a strongly tortuous ER, numerous mitochondria and Golgi apparatus with two types of vesicles. In the cellular cortex, glial cells are much less numerous than the neurons and they are located preferably at the border between perikarya and neuropil. Furthermore, they send many branching shoots between the surrounding neuron perikarya and the axons. The glial cytoplasmic matrix appears more electrodense than that of the neurons. Only few ribosomes are attached to the membranes of the ER; the vast majorities are free. In the perikarya of the glial cells, mitochondria, multi-vesicular bodies, various vacuoles and vesicles are present. The essential elements of the hemolymph-brain barrier are the endothelial cells with their tight junctions. The cytoplasm contains various vesicles and mitochondria. However, two other cell types are present, the pericytes and the astrocytes, which are of great importance for the function of the hemolymph-brain barrier. The cell-cell interactions between endothelial cells, pericytes and astrocytes are as close as no other cells.

Mitochondria-Derived H2O2 Promotes Symmetry Breaking of the C.elegans Zygote.

Symmetry breaking is an essential step in cell differentiation and early embryonic development. However, the molecular cues that trigger symmetry breaking remain largely unknown. Here, we show that mitochondrial H2O2 acts as a symmetry-breaking cue in the C.elegans zygote. We find that symmetry breaking is marked by a local H2O2 increase and coincides with a relocation of mitochondria to the cell cortex. Lowering endogenous H2O2 levels delays the onset of symmetry breaking, while artificially targeting mitochondria to the cellular cortex using a light-induced heterodimerization technique is sufficient to initiate symmetry breaking in a H2O2-dependent manner. In wild-type development, both sperm and maternal mitochondria contribute to symmetry breaking. Our findings reveal that mitochondrial H2O2-signaling promotes the onset of polarization, a fundamental process in development and cell differentiation, and this is achieved by both mitochondrial redistribution and differential H2O2-production.