Intracellular Polarization Research Articles

Deep learning-based cytoskeleton segmentation for accurate high-throughput measurement of cytoskeleton density.

Microscopic analyses of cytoskeleton organization are crucial for understanding various cellular activities, including cell proliferation and environmental responses in plants. Traditionally, assessments of cytoskeleton dynamics have been qualitative, relying on microscopy-assisted visual inspection. However, the transition to quantitative digital microscopy has introduced new technical challenges, with segmentation of cytoskeleton structures proving particularly demanding. In this study, we examined the utility of a deep learning-based segmentation method for accurate quantitative evaluation of cytoskeleton organization using confocal microscopic images of the cortical microtubules in tobacco BY-2 cells. The results showed that, although conventional methods sufficed for measurement of cytoskeleton angles and parallelness, the deep learning-based method significantly improved the accuracy of density measurements. To assess the versatility of the method, we extended our analysis to physiologically significant models in the context of changes in cytoskeleton density, namely Arabidopsis thaliana guard cells and zygotes. The deep learning-based method successfully improved the accuracy of cytoskeleton density measurements for quantitative evaluations of physiological changes in both stomatal movement in guard cells and intracellular polarization in elongating zygotes, confirming its utility in these applications. The results demonstrate the effectiveness of deep learning-based segmentation in providing precise and high-throughput measurements of cytoskeleton density, and has the potential to automate and expedite analyses of large-scale image datasets.

Observation of polarity changes in Sjogren's syndrome mice using a targeting lysosomes and ratiometric fluorescent probe

Utilizing non-invasive, real-time dynamic imaging and high-resolution detection tools to track polarity changes in Sjögren's syndrome (SS) contributes to a better understanding of the disease progression. Herein, a ratiometric polarity-sensitive fluorescent probe (DIM) was designed and synthesized, DIM consisted of dicyanoisophorone as the fluorophore and morpholine moiety as lysosome targeting. DIM showed a ratiometric response to polarity and high selectivity (unaffected by viscosity, pH, ROS, RNS, etc.), offering a more accurate analysis of intracellular polarity through a built-in internal reference calibration. The polarity abnormality of submandibular glands in non-obese diabetic (NOD) mice was revealed and verified by in vivo ratiometric fluorescence imaging of DIM, suggesting that fluorescent probe have great potential in the diagnosis of salivary gland abnormalities.

The remnant of the midbody as a cellular signaling mechanism

Cell signaling mechanisms are the basis for intercellular integration and regulation of proliferation and differentiation processes at the systemic level. One of the most plausible ways to control cell-to-cell interaction and targeted distribution of genetic information is for cells to use their own structures that are formed during mitosis and carry RNA-dependent signaling molecules that affect the mechanisms of control of intercellular interaction, cell proliferation and differentiation. The midbody remnant is a microtubule-rich structure that forms between dividing cells in the last stages of cytokinesis. Previously, it was thought to be only a temporary structure of the intercellular bridge during cytokinesis, which served to connect two future daughter cells. This structure is a key regulator of abscission and functions as a signaling platform that coordinates the cytoskeleton and endosomal dynamics during the terminal stages of cell division. The midbody is a subcellular structure that is formed during cell division, during penetration into the cleavage sulcus, when the microtubules of the central spindle are compacted and cross-linked by a thin intracellular bridge connecting the two daughter cells. The midbody plays a key role in organizing cytokinesis by recruiting a variety of mitotic kinases such as Aurora B and Plk1, as well as sulcus endosomes containing Rab11/FIP3, the membrane-rupturing ESCRT complex and the microtubule-rupturing enzyme spastin, all of which are responsible for mediated rupture during the later stages of cytokinesis. The midbodies can serve as extracellular and intracellular polarity signals during early embryogenesis, as well as during epithelialization and polarization of neurons. The molecular mechanism that governs the positioning of the middle body and how it transmits signals to neurons during differentiation or epithelium remains unknown. Importantly, the remains of the middle bodies can also function as intracellular signaling scaffolds that regulate proliferation and fate postmitotic cells. Since these structures can be released outside cells and taken up by other non-mitotic cells, it is suggested that they may function as vehicles for alternative transmission of complex sets of signaling molecules and/or receptors between cells, thus profoundly affecting signaling in general.

A NIR Dual-Channel Fluorescent Probe for Fluctuations of Intracellular Polarity and H2O2 and Its Applications for the Visualization of Inflammation and Ferroptosis.

Hydrogen peroxide and polarity are closely related to many physiological activities and pathological processes. However, near-infrared fluorescent probes that are sensitive to both H2O2 and polarity are still scarce. Herein, we developed the first dual-channel near-infrared fluorescent probe NBO, with an AIE effect, enabling simultaneous monitoring of H2O2 and polarity. The probe presented high sensitivity, high selectivity, and low detection limit for H2O2. It also had high sensitivity to polarity, independent of pH and viscosity, with large Stokes shifts, good photostability, and low cytotoxicity. Moreover, NBO was able to detect both endogenous and exogenous H2O2 as well as polarity fluctuations in vivo as a method to effectively differentiate between cancer cells and normal cells. Importantly, it also could monitor the therapeutic effects of drugs in inflammation and iron-dead cells and mice. Based on NIR emission, NBO could be used as an imaging tool and a way to evaluate the therapeutic effect of drugs for inflammation and ferroptosis.

A lipid droplets-targetable fluorescent probe for polarity detection in cells of iron death, inflammation and fatty liver tissue

Abnormal accumulation and metabolism of lipid droplets can lead to a variety of diseases. Polarity, a key parameter of the microenvironment, is closely associated with many diseases and dysfunctions in the body. It is important to elucidate the relationship between the physiological activity of lipid droplets (LDs) and the polarity of the microenvironment. In this work, based on push-pull mechanism, a fluorescent probe (E)-3-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)-1-(2-hydroxyphenyl)prop‑2-en-1-one (PPTH) with aggregation-induced emission (AIE) properties for the detection of polarity changes in cells was synthesized. PPTH not only visualize intracellular polarity fluctuation of iron death and inflammation but also distinguish between normal and fatty liver tissue.

Persistent cell contacts enable E-cadherin/HMR-1- and PAR-3-based symmetry breaking within a developing C. elegans epithelium

Tissue-wide patterning is essential to multicellular development, requiring cells to individually generate polarity axes and coordinate them in space and time with neighbors. Using the C.elegans intestinal epithelium, we identified a patterning mechanism that is informed by cell contact lifetime asymmetry and executed via the scaffolding protein PAR-3 and the transmembrane protein E-cadherin/HMR-1. Intestinal cells break symmetry as PAR-3 and HMR-1 recruit apical determinants into punctate "local polarity complexes" (LPCs) at homotypic contacts. LPCs undergo an HMR-1-based migration to a common midline, thereby establishing tissue-wide polarity. Thus, symmetry breaking results from PAR-3-dependent intracellular polarization coupled to HMR-1-based tissue-level communication, which occurs through a non-adhesive signaling role for HMR-1. Differential lifetimes between homotypic and heterotypic cell contacts are created by neighbor exchanges and oriented divisions, patterning where LPCs perdure and thereby breaking symmetry. These cues offer a logical and likely conserved framework for how epithelia without obvious molecular asymmetries can polarize.

A multidimensional quantitative fluorescent probe for high-fidelity monitoring of the polarity change in lysosomes during autophagy

The current majority of fluorescent probes are primarily based on fluorescence imaging which relies on single fluorescence emission intensity, and the complex intracellular biological environment dramatically interferes with the accuracy of the assay. Fluorescence lifetime imaging (FLIM) can take advantage of the long-life characteristics of fluorescent probes for background-free optical analysis imaging. However, it places higher demands on designing and preparing of optical probes. An environment-sensitive near-infrared (NIR) probe, TPA-DCPP NPs, was prepared to achieve multidimensional quantification (confocal fluorescence intensity, in situ emission, and lifetime imaging) of intracellular polarity enabling accurate cancer cell discrimination and autophagy monitoring, which is expected to provide ideas for developing probes realizing detect targets precisely in complex biosystems.

Reciprocal intra- and extra-cellular polarity enables deep mechanosensing through layered matrices

Adherent cells migrate on layered tissue interfaces to drive morphogenesis, wound healing, and tumor invasion. Although stiffer surfaces are known to enhance cell migration, it remains unclear whether cells sense basal stiff environments buried under softer, fibrous matrix. Using layered collagen-polyacrylamide gel systems, we unveil a migration phenotype driven by cell-matrix polarity. Here, cancer (but not normal) cells with stiff base matrix generate stable protrusions, faster migration, and greater collagen deformation because of "depth mechanosensing" through the top collagen layer. Cancer cell protrusions with front-rear polarity produce polarized collagen stiffening and deformations. Disruption of either extracellular or intracellular polarity via collagen crosslinking, laser ablation, or Arp2/3 inhibition independently abrogates depth-mechanosensitive migration of cancer cells. Our experimental findings, validated by lattice-based energy minimization modeling, present a cell migration mechanism whereby polarized cellular protrusions and contractility are reciprocated by mechanical extracellular polarity, culminating in a cell-type-dependent ability to mechanosense through matrix layers.

Engineering hydrophilic/hydrophobic moieties of ratiometric fluorescence probe to visualize whole cytoplasmic polarity

Intracellular polarity is an essential parameter to indicate the property of microenvironment in the whole cell. The polarity is closely related to various physiological processes in different organelles and their changes in real time depending on the physiological/pathological activity. Visualization of whole cytoplasmic polarity is essential to understand the multi-organelle related pathological and biological phenomena. However, most of the reported single-organelle targeted fluorescent materials can only detect specific intracellular regions and normally with monotonic fluorescence changes. Herein, we engineered a polarity-sensitive probe 1P2N that install a cationic side alkyl chain on a push–pull type of pyrenyl-pyridium dye for whole cytoplasmic polarity visualization. Especially, this probe emits blue-green fluorescence from pyrene moiety in high-polar hydrophilic cytoplasm, while emits red fluorescence from intramolecular charge transfer process in low-polar lipidic organelles. The dual-emission response of 1P2N to polarity provides the basis for its ratiometric fluorescence monitoring of polarity changes in the whole cytoplasm under different physiological conditions. The excellent polarity-sensitive properties of probe 1P2N make it a useful tool for understanding polarity-related processes in the cytoplasmic microenvironment.

A red-emitting thiophene-modified BODIPY probe for fluorescence lifetime-based polarity imaging of lipid droplets in living cells.

Intracellular polarity in lipid droplets as well as other organelles may provide useful knowledge about various processes taking place in live cells. Therefore, small fluorophores capable of visualising polarity are undergoing rapid development. In this paper, we report new red-emitting polarity sensitive BODIPY probes that can distinguish between liquid-ordered and liquid-disordered phases and can internalise into lipid droplets of live cells. Our reported probes sense lipid environment not through solvatochromic shift of the fluorescence spectra but through the change in the fluorescence lifetime of their monoexponential decays. This makes them convenient for fluorescence lifetime imaging microscopy. The probes were synthesised by modifying viscosity-sensitive meso-phenyl BODIPY with electron-donating 2-thienyl moieties at the α- and β-positions, significantly red-shifting absorption and fluorescence spectra of the dyes and improving sensitivity to polarity, while suppressing viscosity dependence. Finally, a novel probe - BP OC16 TP2 was suitable for sensing polarity in lipid droplets of live MCF-7 human breast cancer cells. We demonstrated that different chemotherapeutics affected lipid droplet polarity differently: cisplatin had no effect on lipid droplet polarity, whereas paclitaxel, depending on its concentration, either decreased or increased lipid droplet polarity.

Rational Design of a Near-Infrared Ratiometric Probe with a Large Stokes Shift: Visualization of Polarity Abnormalities in Non-Alcoholic Fatty Liver Model Mice.

Tracking liver polarity with noninvasive and dynamic imaging techniques is helpful to better understand the non-alcoholic fatty liver (NAFL). Herein, a novel near-infrared (NIR) fluorescent probe Cy-Mp is constructed using a "symmetry collapse" strategy. The structure modification leads to the conversion of locally excited state fluorescence to charge transfer state fluorescence. Cy-Mp emits at near-infrared (NIR) wavelengths with high photostability as well as a large Stokes shift. Cy-Mp exhibits a ratiometric response to polarity, providing more accurate analysis of intracellular polarity via the built-in internal reference correction. Most importantly, the in vivo studies indicate that Cy-Mp can accumulate in the liver and the decreased polarity in the liver of mice with NAFL is verified by the ratiometric imaging, implying the great potential of Cy-Mp in the diagnosis of NAFL.

A Rhodol-based Fluorescent Probe with a Pair of Hydrophilic and Rotatable Wings for Sensitively Monitoring Intracellular Polarity.

Cell polarity, as a vital intracellular microenvironment characteristic, has immense effects on numerous pathological and biological processes. Therefore, the tracking of polarity variations is highly essential to explore the role and mechanism of the polarity in pathophysiological processes. Herein, we designed and synthesized a novel rhodol-based fluorescent probe (RDS) sensitive to polarity by introducing a bis(2-hydroxyethylthio)methyl group, like a pair of hydrophilic and rotatable wings, into the rhodol skeleton. This unique design makes RDS adopt the colorless and non-fluorescent spirocyclic form in a low-polarity medium while the colored and fluorescent ring-open form is present in a high-polarity system, resulting in a positive-correlation response of fluorescence intension to polarity. Importantly, RDS was successfully applied to monitor the polarity changes in living cells including cancer cells, healthy cells and senescent healthy cells, visualizing that the polarity of cancer cells is lower than that of healthy cells in which the more senescent ones have higher polarity.

Folding for the Immune Synapse: CCT Chaperonin and the Cytoskeleton.

Lymphocytes rearrange their shape, membrane receptors and organelles during cognate contacts with antigen-presenting cells (APCs). Activation of T cells by APCs through pMHC-TCR/CD3 interaction (peptide-major histocompatibility complex-T cell receptor/CD3 complexes) involves different steps that lead to the reorganization of the cytoskeleton and organelles and, eventually, activation of nuclear factors allowing transcription and ultimately, replication and cell division. Both the positioning of the lymphocyte centrosome in close proximity to the APC and the nucleation of a dense microtubule network beneath the plasma membrane from the centrosome support the T cell’s intracellular polarity. Signaling from the TCR is facilitated by this traffic, which constitutes an important pathway for regulation of T cell activation. The coordinated enrichment upon T cell stimulation of the chaperonin CCT (chaperonin-containing tailless complex polypeptide 1; also termed TRiC) and tubulins at the centrosome area support polarized tubulin polymerization and T cell activation. The proteasome is also enriched in the centrosome of activated T cells, providing a mechanism to balance local protein synthesis and degradation. CCT assists the folding of proteins coming from de novo synthesis, therefore favoring mRNA translation. The functional role of this chaperonin in regulating cytoskeletal composition and dynamics at the immune synapse is discussed.

Cell-ECM contact-guided intracellular polarization is mediated via lamin A/C dependent nucleus-cytoskeletal connection

Cell polarization plays a crucial role in dynamic cellular events, such as cell proliferation, differentiation, and directional migration in response to diverse extracellular and intracellular signals. Although it is well known that cell polarization entails highly orchestrated intracellular molecular reorganization, the underlying mechanism of repositioning by intracellular organelles in the presence of multiple stimuli is still unclear. Here, we show that front-rear cell polarization based on the relative positions of nucleus and microtubule organizing center is precisely controlled by mechanical interactions including cellular adhesion to extracellular matrix and nucleus-cytoskeletal connections. By modulating the size and distribution of fibronectin-coated adhesive spots located in the polarized cell shape mimicking micropatterns, we monitored the alterations in cell polarity. We found that the localization of individual adhesive spots is more dominant than the cell shape itself to induce intracellular polarization. Further, the degree of cell polarization was diminished significantly by disrupting nuclear lamin A/C. We further confirm that geometrical cue-guided intracellular polarization determines directional cell migration via local activation of Cdc42. These findings provide novel insights into the role of nucleus-cytoskeletal connections in single cell polarization under a combination of physical, molecular, and genetic cues, where lamin A/C acts as a critical molecular mediator in ECM sensing and signal transduction via nucleus-cytoskeletal connection.

Building Accurate Intracellular Polarity Maps through Multiparametric Microscopy.

The precise knowledge of intracellular polarity, a physiological parameter that involves complex and intertwined intracellular mechanisms, may be relevant in the study of important diseases like cancer or Alzheimer’s. In this technical note, we illustrate our recently developed, accurate method for obtaining intracellular polarity maps employing potent fluorescence microscopy techniques. Our method is based on the selection of appropriate luminescent probes, in which several emission properties vary with microenvironment polarity, specifically spectral shifts and luminescence lifetime. A multilinear calibration is performed, correlating polarity vs. spectral shift vs. luminescence lifetime, to generate a powerful and error-free 3D space for reliable interpolation of microscopy data. Multidimensional luminescence microscopy is then used to obtain simultaneously spectral shift and luminescence lifetime images, which are then interpolated in the 3D calibration space, resulting in accurate, quantitative polarity maps.

Simultaneous monitoring of polarity changes of lipid droplets and lysosomes with two-photon fluorescent probes

Intracellular polarity is an essential feature of cell physiological state and abnormal polarity changes of various organelles are related to many diseases. Thus, monitoring of polarity changes of multiple subcellular in living cells contributes to understanding different physiological and pathological processes more accurately. However, most of the previous reports on polarity probes mainly monitored the polarity of a single organelle. Therefore, we designed and synthesized two unique polarity-sensitive fluorescent probes LDs-TPFP and Lyso-TPFP, which can be selectively located in lipid droplets (LDs) and lysosomes respectively, to obtain more subcellular information in living cells. Thanks to the strong intramolecular-charge-transfer (ICT) characteristics of probes, the fluorescence intensity and emission wavelength would change with the polarity of the surroundings of cells. Moreover, LDs-TPFP and Lyso-TPFP exhibits large Stokes shift and excellent biocompatibility. Through fluorescence imaging, the probes can effectively distinguish normal cells from cancer cells. In addition, the results of two-photon confocal fluorescence imaging indicated that LDs and lysosomes have discrepant polarity change behaviors under different physiological conditions.

Mechanical Cell-to-Cell Interactions as a Regulator of Topological Defects in Planar Cell Polarity Patterns in Epithelial Tissues

A precise structural organization of epithelial cells is needed for the proper functioning and development of different tissues. The epithelial cell packing mechanism is associated with mechanical interactions between cells that place the tissue in a state of the lowest mechanical energy. In addition, the planar cell polarity has been recognized as another important mechanism for the epithelial organization that ensures the orientational ordering of the cells. Planar cell polarity is a consequence of an asymmetric distribution of certain transmembrane proteins which is driven by specific intracellular signaling pathways. Mutations and other disruptions of these pathways were found to cause an impaired cytoarchitecture of the epithelium layer. Mutant cells with disrupted activities of signaling proteins basically represent topological defects, which can lead to dysfunctions in tissue operation. Motivated by the fact that regional variations of bond tensions were found in the vicinity of mutant cells, we implement a computational model that combines the tissue development processes following the concept of mechanical energy minimization and the intracellular polarization. Our results reveal that a decrease in mechanical interactions between normal and mutant epithelium cells represents a conceivable regulatory mechanism that diminishes the impact of topological defects caused by mutant cells.

Multiscale modelling of motility wave propagation in cell migration

The collective motion of cell monolayers within a tissue is a fundamental biological process that occurs during tissue formation, wound healing, cancerous invasion, and viral infection. Experiments have shown that at the onset of migration, the motility is self-generated as a polarisation wave starting from the leading edge of the monolayer and progressively propagates into the bulk. However, it is unclear how the propagation of this motility wave is influenced by cellular properties. Here, we investigate this question using a computational model based on the Potts model coupled to the dynamics of intracellular polarisation. The model captures the propagation of the polarisation wave and suggests that the cells cortex can regulate the migration modes: strongly contractile cells may depolarise the monolayer, whereas less contractile cells can form swirling movement. Cortical contractility is further found to limit the cells motility, which (i) decelerates the wave speed and the leading edge progression, and (ii) destabilises the leading edge. Together, our model describes how different mechanical properties of cells can contribute to the regulation of collective cell migration.

Pattern formation in a coupled membrane-bulk reaction-diffusion model for intracellular polarization and oscillations

Reaction-diffusion systems have been widely used to study spatio-temporal phenomena in cell biology, such as cell polarization. Coupled bulk-surface models naturally include compartmentalization of cytosolic and membrane-bound polarity molecules. Here we study the distribution of the polarity protein Cdc42 in a mass-conserved membrane-bulk model, and explore the effects of diffusion and spatial dimensionality on spatio-temporal pattern formation. We first analyze a one-dimensional (1-D) model for Cdc42 oscillations in fission yeast, consisting of two diffusion equations in the bulk domain coupled to nonlinear ODEs for binding kinetics at each end of the cell. In 1-D, our analysis reveals the existence of symmetric and asymmetric steady states, as well as anti-phase relaxation oscillations typical of slow-fast systems. We then extend our analysis to a two-dimensional (2-D) model with circular bulk geometry, for which species can either diffuse inside the cell or become bound to the membrane and undergo a nonlinear reaction-diffusion process. We also consider a nonlocal system of PDEs approximating the dynamics of the 2-D membrane-bulk model in the limit of fast bulk diffusion. In all three model variants we find that mass conservation selects perturbations of spatial modes that simply redistribute mass. In 1-D, only anti-phase oscillations between the two ends of the cell can occur, and in-phase oscillations are excluded. In higher dimensions, no radially symmetric oscillations are observed. Instead, the only instabilities are symmetry-breaking, either corresponding to stationary Turing instabilities, leading to the formation of stationary patterns, or to oscillatory Turing instabilities, leading to traveling and standing waves. Codimension-two Bogdanov–Takens bifurcations occur when the two distinct instabilities coincide, causing traveling waves to slow down and to eventually become stationary patterns. Our work clarifies the effect of geometry and dimensionality on behaviors observed in mass-conserved cell polarity models.

Orthogonal cell polarity imaging by multiparametric fluorescence microscopy

The cellular microenvironment is a complex medium due to high concentrations of proteins and an intertwined framework of cellular organelles. In particular, cellular micro-polarity controls several biological processes, since it modulates hydrophobic/hydrophilic interactions and, hence, recognition, signalling and binding events.In this work, we have developed an unprecedented methodology to construct accurate environment polarity images using multiparametric fluorescence microscopy, via a multi-linear calibration of orthogonal parameters: the fluorescence lifetime and the spectral shift of a series of solvatochromic dyes. For this approach, we have synthesized and fully characterized N-substituted 2-methoxy-9-acridone dyes as suitable bioimaging polarity probes. However, to fully comprehend the complex links between microenvironment polarity and the dyes’ properties, we have endeavoured a multidisciplinary approach, in which we have studied the photophysics of our fluorophores using spectroscopic tools and state-of-the-art computational chemistry. This profound knowledge permitted to use these dyes as intracellular polarity probes, quantitatively and robustly probing the microenvironment of different cellular compartments.Our new methodology may pave the way to further developments in accurate sensing of cellular microenvironment parameters.