•We propose a novel Faster Mean-shift algorithm, which accelerates the embedding clustering based one-stage holistic cell instance segmentation and tracking.•The proposed Faster Mean-shift achieved ...7-10 times speed-up compared with the state-of-the-art embedding based cell instance segmentation and tracking algorithm.•We propose the new online seed optimization policy (OSOP) and early stopping strategy to achieve the best computational speed with optimized memory consumption, compared with previous GPU-based benchmarks.•Comprehensive simulations for in-depth theoretical analysis, as well as empirical validations on four ISBI cell tracking challenge data-set have been performed in this study.
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Recently, single-stage embedding based deep learning algorithms gain increasing attention in cell segmentation and tracking. Compared with the traditional “segment-then-associate” two-stage approach, a single-stage algorithm not only simultaneously achieves consistent instance cell segmentation and tracking but also gains superior performance when distinguishing ambiguous pixels on boundaries and overlaps. However, the deployment of an embedding based algorithm is restricted by slow inference speed (e.g., ≈1–2 min per frame). In this study, we propose a novel Faster Mean-shift algorithm, which tackles the computational bottleneck of embedding based cell segmentation and tracking. Different from previous GPU-accelerated fast mean-shift algorithms, a new online seed optimization policy (OSOP) is introduced to adaptively determine the minimal number of seeds, accelerate computation, and save GPU memory. With both embedding simulation and empirical validation via the four cohorts from the ISBI cell tracking challenge, the proposed Faster Mean-shift algorithm achieved 7–10 times speedup compared to the state-of-the-art embedding based cell instance segmentation and tracking algorithm. Our Faster Mean-shift algorithm also achieved the highest computational speed compared to other GPU benchmarks with optimized memory consumption. The Faster Mean-shift is a plug-and-play model, which can be employed on other pixel embedding based clustering inference for medical image analysis. (Plug-and-play model is publicly available: https://github.com/masqm/Faster-Mean-Shift)
Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis 1–3. Mutations in ...NMII are associated with a number of diseases due to failures in cell-cell adhesion 4–8. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400–600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.
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► NMII within epithelial junctions assembles into a chain of muscle-like sarcomeres. ► Sarcomeres are paired across the junction to form transcellular contractile units. ► NMII isoforms show differential expression across heterotypic cell junctions. ► Coordinated sarcomere contractility modulates apical cell shape and tissue geometry
Mechanosensitive ion channels at stereocilia tips mediate mechanoelectrical transduction (MET) in inner ear sensory hair cells. Transmembrane channel-like 1 and 2 (TMC1 and TMC2) are essential for ...MET and are hypothesized to be components of the MET complex, but evidence for their predicted spatiotemporal localization in stereocilia is lacking. Here, we determine the stereocilia localization of the TMC proteins in mice expressing TMC1-mCherry and TMC2-AcGFP. Functionality of the tagged proteins was verified by transgenic rescue of MET currents and hearing in Tmc1Δ/Δ;Tmc2Δ/Δ mice. TMC1-mCherry and TMC2-AcGFP localize along the length of immature stereocilia. However, as hair cells develop, the two proteins localize predominantly to stereocilia tips. Both TMCs are absent from the tips of the tallest stereocilia, where MET activity is not detectable. This distribution was confirmed for the endogenous proteins by immunofluorescence. These data are consistent with TMC1 and TMC2 being components of the stereocilia MET channel complex.
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•TMC1 and TMC2 localize at the site of hair cell mechanotransduction•Expression of TMC2 in cochlea is limited to developing stereocilia bundles•TMC1 is an essential component of the mechanotransduction channel complex
The molecular makeup of hair cell mechanoelectrical transduction (MET) complex remains elusive. Kurima et al. show that fluorophore-tagged TMC1/2 localize to MET sites at stereocilia tips and that their expression in TMC1/2-null mice rescues MET. These findings support the model that TMC1/2 are part of the MET complex.
Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the ...diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.
Transporting epithelial cells generate arrays of microvilli, known as a brush border, to enhance functional capacity. To understand brush border formation, we used live cell imaging to visualize ...apical remodeling early in this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at ∼0.2 μm/min. Perturbation with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed ends of core bundles, which in turn is linked to robust treadmilling of these structures. Actin regulatory factors IRTKS and EPS8 localize to the barbed ends of motile microvilli, where they control the kinetics and nature of movement. As the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately clustering and consolidation into higher-order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.
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•Microvilli exhibit persistent, active motility driven by actin assembly•Microvillar F-actin cores treadmill during motility•Barbed-end binding factors regulate microvillar motility•Motility promotes intermicrovillar collisions, adhesion, and cluster formation
Transporting epithelial cells assemble extensive apical arrays of microvilli, known as brush borders. Meenderink et al. use live cell imaging to reveal that nascent microvilli translocate across the cell surface. Cells harness this motility to consolidate microvilli into clusters and tightly packed arrays during differentiation.
Transporting epithelial cells, like those that line the intestinal tract, are specialized for solute processing and uptake. One defining feature is the brush border, an array of microvilli that ...serves to amplify apical membrane surface area and increase functional capacity. During differentiation, upon exit from stem-cell-containing crypts, enterocytes build thousands of microvilli, each supported by a parallel bundle of actin filaments several microns in length. Given the high concentration of actin residing in mature brush borders, we sought to determine whether enterocytes were resource (i.e., actin monomer) limited in assembling this domain. To examine this possibility, we inhibited Arp2/3, the ubiquitous branched actin nucleator, to increase G-actin availability during brush border assembly. In native intestinal tissues, Arp2/3 inhibition led to increased microvilli length on the surface of crypt, but not villus, enterocytes. In a cell culture model of brush border assembly, Arp2/3 inhibition accelerated the growth and increased the length of microvilli; it also led to a redistribution of F-actin from cortical lateral networks into the brush border. Effects on brush border growth were rescued by treatment with the G-actin sequestering drug, latrunculin A. G-actin binding protein, profilin-1, colocalized in the terminal web with G-actin, and knockdown of this factor compromised brush border growth in a concentration-dependent manner. Finally, the acceleration in brush border assembly induced by Arp2/3 inhibition was abrogated by profilin-1 knockdown. Thus, brush border assembly is limited by G-actin availability, and profilin-1 directs unallocated actin monomers into microvillar core bundles during enterocyte differentiation.
•Inhibition of Arp2/3 accelerates growth of microvilli in vivo and in vitro•Inhibition of Arp2/3 redistributes F-actin from the cortex into microvilli•Profilin-1 is required for normal brush border assembly•Acceleration of microvilli growth upon inhibition of Arp2/3 requires profilin-1
Faust et al. show that the rate of brush border assembly is limited by G-actin availability and that profilin-1 directs unallocated G-actin into growing microvilli during enterocyte differentiation. These findings offer a framework for understanding how epithelial cells cope with the demands of building elaborate apical specializations.
Abstract Background aims Multipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive ...properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed. Methods Human bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red–positive adipocytes after automated image acquisition. Results MSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences. Conclusions We measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.
To maximize solute transport, epithelial cells build an apical “brush border,” where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion ...complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.
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•Nascent microvilli accumulate at cell margins early in epithelial differentiation•Marginal microvilli exhibit reduced motility and a more vertical orientation•Marginal microvilli from neighboring cells are linked by complexes of CDHR2/CDHR5•Transjunctional CDHR2/CDHR5 links are long-lived and drive microvilli accumulation
Cencer et al. demonstrate that microvilli accumulation during the early stages of epithelial differentiation is driven by the formation of transjunctional adhesion complexes of CDHR2 and CDHR5, which link protrusions that extend from neighboring cells. Transjunctional adhesion stabilizes microvilli at these sites and ultimately drives their accumulation as differentiation progresses.
Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed ...microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:
Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae ...accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca(2+)-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins. DOI:http://dx.doi.org/10.7554/eLife.00926.001.
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