Abl family nonreceptor tyrosine kinases regulate changes in cell shape and migration. Abl2 localizes to dynamic actin-rich protrusions, such as lamellipodia in fibroblasts and dendritic spines in neurons. Abl2 interactions with cortactin, an actin filament stabilizer, are crucial for the formation and stability of actin-rich structures, but Abl2:cortactin-positive structures have not been characterized with high spatiotemporal resolution in cells. Using total internal reflection fluorescence microscopy, we demonstrate that Abl2 colocalizes with cortactin at wave-like structures within lamellum and lamellipodium tips. Abl2 and cortactin within waves are focal and transient, extend to the outer edge of lamella, and serve as the base for lamellipodia protrusions. Abl2-positive foci colocalize with integrin β3 and paxillin, adhesive markers of the lamellum-lamellipodium interface. Cortactin-positive waves still form in Abl2 knockout cells, but the lamellipodium size is significantly reduced. This deficiency is restored following Abl2 reexpression. Complementation analyses revealed that the Abl2 C-terminal half, which contains domains that bind actin and microtubules, is necessary and sufficient for recruitment to the wave-like structures and to support normal lamellipodium size, while the kinase domain-containing N-terminal half does not impact lamellipodium size. Together, this work demonstrates that Abl2 is recruited with cortactin to actin waves through cytoskeletal interactions to promote lamellipodium extension.

Comparative studies of subspecies under different ecological environments offer insights into intraspecies evolutionary adaptive mechanisms. Golden snub-nosed monkeys (Rhinopithecus roxellana) include three subspecies in China classified mainly by their morphological variations: R. r. roxellana (Sichuan and Gansu province), R. r. qinlingensis (Shaanxi province) and R. r. hubeiensis (Hubei province). These three subspecies live in three isolated area with different environments. Past works focused on the last two subspecies, but little information of habitat and behaviors of the nominated subspecies (R. r. roxellana) is available to date. We conducted a two-year study on the diet, activity budget, home range and social organization of 4 herds of R. r. roxellana, based on a total of 106 days' observation in Laohegou (LHG) Nature Reserve, Sichuan province. By using scan sampling method, our results suggest that the R. r roxellana feeds predominantly on leaves (77.5%), and spends more time feeding (40.0%) and resting (27.0%) while compared to the other two subspecies. Kernel Density Estimation Method based on GPS technology confirms that R. r roxellana has relatively larger home ranges (49.1 km2). The unit size (8.3 ± 3.5 individuals) of R. r roxellana is also smaller. Therefore, it is possible that differences in food availability in relation to habitats have important impacts on the feeding strategy and social system of the golden snub-nosed monkey. These results provide data to further explore intraspecific adaptations of living primates.

Dendritic RNAs are localized and translated in RNA granules. Here we use single-molecule imaging to count the number of RNA molecules in each granule and to record translation output from each granule using Venus fluorescent protein as a reporter. For RNAs encoding activity-regulated cytoskeletal-associated protein (ARC) or fragile X mental retardation protein (FMRP), translation events are spatially clustered near individual granules, and translational output from individual granules is either sporadic or bursty. The probability of bursty translation is greater for Venus-FMRP RNA than for Venus-ARC RNA and is increased in Fmr1-knockout neurons compared to wild-type neurons. Dihydroxyphenylglycine (DHPG) increases the rate of sporadic translation and decreases bursty translation for Venus-FMRP and Venus-ARC RNAs. Single-molecule imaging of translation in individual granules provides new insight into molecular, spatial, and temporal regulation of translation in granules.

Automated cell segmentation from optical microscopy images is usually the first step in the pipeline of single-cell analysis. Recently, deep-learning based algorithms have shown superior performances for the cell segmentation tasks. However, a disadvantage of deep-learning is the requirement for a large amount of fully annotated training data, which is costly to generate. Weakly-supervised and self-supervised learning is an active research area, but often the model accuracy is inversely correlated with the amount of annotation information provided. Here we focus on a specific subtype of weak annotations, which can be generated programmably from experimental data, thus allowing for more annotation information content without sacrificing the annotation speed. We designed a new model architecture for end-to-end training using such incomplete annotations. We have benchmarked our method on a variety of publicly available datasets, covering both fluorescence and bright-field imaging modality. We additionally tested our method on a microscopy dataset generated by us, using machine-generated annotations. The results demonstrated that our models trained under weak supervision can achieve segmentation accuracy competitive to, and in some cases, surpassing, state-of-the-art models trained under full supervision. Therefore, our method can be a practical alternative to the established full-supervision methods.

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

Using photoactivatable fluorescent protein as an intracellular protein label for single-molecule tracking offers several advantages over the traditional methods. Here we demonstrate the technique of photoactivation single-molecule tracking by investigating the mobility dynamics of intracellular FtsZ protein molecules in live Escherichia coli cells. FtsZ is a prokaryotic cytoskeleton protein (a homolog of tubulin) and plays important roles in cytokinesis. We demonstrate two heterogeneous subpopulations of FtsZ molecules with distinct diffusional dynamics. The FtsZ molecules forming the Z-rings near the center of the cell were mostly stationary, consistent with the assumption that they are within polymeric filamentous structures. The rest of the FtsZ molecules, on the other hand, undergo Brownian motion spanning the whole cell length. Surprisingly, the diffusion of FtsZ is spatially restricted to helical-shaped regions, implying an energy barrier for free diffusion. Consistently, the measured mean-square displacements of FtsZ showed anomalous diffusion characteristics. These results demonstrated the feasibility and advantages of photoactivation single-molecule tracking, and suggested new levels of complexity in the prokaryotic membrane organization.

The aim of the present study was to investigate the association between Ki-67 expression and radiomics features of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in patients with invasive breast cancer. A total of 53 cases with low-Ki-67 expression (Ki-67 proliferation index <14%) and 106 cases with high-Ki-67 expression (Ki-67 proliferation index >14%) were investigated. A systematic approach was applied that focused on the automated segmentation of lesions and extraction of radiomics features. For each lesion 5 morphology, 4 gray-scale histogram and 6 texture features were obtained, and statistical analyzes were performed to assess the differences in these features between the low- and high-Ki-67 expressions. One morphology metric (area), 3 gray-scale histogram indexes (standard deviation, skewness and kurtosis) and 3 texture features (contrast, homogeneity and inverse differential moment) demonstrated a significant difference (P<0.05), with low-Ki-67 expression lesions tending to be smaller, clearer and heterogeneous when compared with the high-Ki-67 expressed cases. These results may provide a noninvasive means to better understand the proliferation of breast cancer.

Erythropoietin-producing hepatocellular (Eph) receptors regulate a wide array of developmental processes by responding to cell-cell contacts. EphB2 is well-expressed in the brain and known to be important for dendritic spine development, as well as for the maintenance of the synapses, although the mechanisms of these functions have not been fully understood. Here we studied EphB2's functions in hippocampal neurons with an optogenetic approach, which allowed us to specify spatial regions of signal activation and monitor in real-time the consequences of signal activation. We designed and constructed OptoEphB2, a genetically encoded photoactivatable EphB2. Photoactivation of OptoEphB2 in fibroblast cells induced receptor phosphorylation and resulted in cell rounding ------- a well-known cellular response to EphB2 activation. In contrast, local activation of OptoEphb2 in dendrites of hippocampal neurons induces rapid actin polymerization, resulting dynamic dendritic filopodial growth. Inhibition of Rac1 and CDC42 did not abolish OptoEphB2-induced actin polymerization. Instead, we identified Abelson tyrosine-protein kinase 2 (Abl2/Arg) as a necessary effector in OptoEphB2-induced filopodia growth in dendrites. These findings provided new mechanistic insight into EphB2's role in neural development and demonstrated the advantage of OptoEphB as a new tool for studying EphB signaling.

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching a large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore, in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.

Dendritic spines are small protrusions that receive synaptic signals in neuronal networks. The actin cytoskeleton plays a key role in regulating spine morphogenesis, as well as in the function of synapses. Here we report the first quantitative measurement of F-actin retrograde flow rate in dendritic filopodia, the precursor of dendritic spines, and in newly formed spines, using a technique based on photoactivation localization microscopy. We found a fast F-actin retrograde flow in the dendritic filopodia but not in the spine necks. The quantification of F-actin flow rates, combined with fluorescence recovery after photobleaching measurements, allowed for a full quantification of spatially resolved kinetic rates of actin turnover, which was not previously feasible. Furthermore we provide evidences that myosin II regulates the actin flow in dendritic filopodia and translocates from the base to the tip of the protrusion upon spine formation. Rac1 inhibition led to mislocalization of myosin II, as well as to disruption of the F-actin flow. These results provide advances in the quantitative understanding of F-actin remodeling during spine formation.

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)-based single-molecule tracking technique to analyze F-actin movements with approximately 30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of approximately 138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

Dendritic filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the filopodia is crucial for synaptogenesis, but the underlying mechanisms are poorly understood. To study filopodial motility, we collected and analyzed image data on filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity, and substrate adhesion gives rise to various filopodial behaviors. We formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility, and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic filopodia dynamics.

Methotrexate (MTX) is an antifolate agent which is widely used in clinic for treating malignancies, rheumatoid arthritis and ectopic pregnancy. As reported, MTX has side effects on gastrointestinal system, nervous system and reproductive system, while its potential damages on oocyte quality are still unclear. It is known that oocyte quality is essential for healthy conception and the forthcoming embryo development. Thus, this work studied the effects of MTX on the oocyte quality.

Abundant nanostructures have been constantly found in various foods, like vinegar, tea, coffee, and milk. However, these structures largely remain unexplored and even been eliminated for stability reasons in food industry. Here we report the isolation, characterization, and antioxidant activities of food nanoparticles (NPs) carrying polyphenols from Chinese rice vinegar. Using a gel-chromatography-based isolation protocol, the vinegar was separated into three major fractions. They were identified as spherical NPs (P1), lollipop-like NPs (P2) and spherical microparticles (P3) with average hydrodynamic diameter of 210, 245,1643 nm, separately. The former two fractions accounted for the major parts of dry matter in the vinegar. The P1-NPs fraction was composed of proteins, carbohydrates, and a high number of polyphenols (15 wt%), demonstrated potent antioxidant activity as determined by ABTS and ORAC assays. Moreover, they effectively quenched peroxyl free radicals in peritoneal macrophages and promoted cellular growth. The P2 fraction contained majority of organic acids, esters and mineral elements of the vinegar. It demonstrated the NPs are bioactive units of the rice vinegar, inspiring the development of novel functional nanomaterials with nutraceutical and pharmaceutical applications.

Background: Aconiti Lateralis Radix Praeparata, also known as Fuzi in Chinese, has been used in Traditional Chinese Medicine for more than 2,000 years. In recent years, some traditional herbal compounds containing Fuzi have achieved positive clinical results in tumor treatment. And the polysaccharide isolated from Fuzi has attracted much attention as a potential immunomodulator. However, its immunomodulatory mechanism remains to be further studied. Aim of the study. Fuzi neutral polysaccharide (FNPS) and cyclophosphamide (CTX) were combined to treat Hepatoma 22 (H22) tumor-bearing mice, and its mechanism of ameliorating immunosuppression caused by CTX was studied. Methods: FNPS was isolated and purified. The molecular weight, functional groups, monosaccharide composition, and apparent morphology were characterized by gel permeation chromatography, Fourier transform infrared spectrometer, ion chromatography and scanning electron microscope, respectively. Through the analysis of tumor, immune organs, and serum cytokine levels of H22 tumor-bearing mice, the immunomodulatory effect and the protective effect on immunosuppressive mice induced by CTX was evaluated. And the immunomodulatory activity of FNPS was further verified by macrophage functional experiments. Results: FNPS was composed of rhamnose, arabinose, galactose, glucose, and mannose in a molar ratio of 0.008:0.017:0.018:0.908:0.048. Its molecular weight was 94 kDa. In vivo experiments showed that 200 mg mL-1 FNPS could alleviate the suppression of immune organs and immune cells caused by CTX treatment, enhance the antitumor effect of CTX, increase the serum levels of Th1 immune-related pro-inflammatory cytokines (IL-1β and IL-6), and decrease Th2 immune-related anti-inflammatory cytokine (IL-10) and tumor-related pro-inflammatory cytokine (TNF-α) in the chemotherapy mice. Functional experiments revealed that 25 μg mL-1 FNPS could promote phagocytosis and proliferation of macrophages. When the concentration reached 50 μg mL-1, it enhanced the migration activity. Conclusion: FNPS has the potential to alleviate the immunosuppressive effect of CTX by activating immune cells and promoting inflammation. It could be used as a potential auxiliary medication for liver cancer treatment.