Time-lapse Fluorescence Imaging Research Articles

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

A Study on the Structure, Optical Properties and Cellular Localization of Novel 1,3-Benzothiazole-Substituted BODIPYs

A library of seven novel 1,3-benzothiazole-substituted BODIPY derivatives with tunable optical properties was synthesized. The new fluorescent dyes exhibited bathochromically shifted absorptions (up to 670 nm) and emissions centered in the red and near-infrared spectral region (up to 677 nm) in comparison to the parent compound 8-phenyl BODIPY (λabs: 499 nm, λemi: 508 nm). (TD)DFT calculations were performed to rationalize the spectroscopic properties of the new dyes. The cellular biodistribution of the new BODIPY dyes, their fluorescence stability and toxicity were investigated in both living and fixed fibroblasts using time-lapse fluorescent imaging and confocal microscopy. Six of the seven new dyes were photostable and non-toxic in vitro at 10 μM concentration. In addition, they efficiently stained the cell membrane, showing diffuse and dotty localization within the cell at low concentrations (1.0 and 0.1 μM). Specifically, dye TC498 was localized in vesicular structures in both live and fixed cells and could be used as a suitable marker in co-staining studies with other commonly used fluorescent probes.

Real-time imaging of individual electropores proves their longevity in cells

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to −400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (“Ca2+ plumes”) faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to −50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

Characterization of Hematopoietic Stem Cell Diversity Using Spatiotemporal Quantitative Phase Imaging

Cells undergo continual transformations, never retaining their original state. Hematopoietic stem cells (HSCs), at the apex of the hematopoietic hierarchy, are not exempt from this dynamic process. In recent years, the advent of single-cell analysis techniques such as scRNAseq has progressively unveiled the heterogeneity within cell populations previously presumed to be homogeneous. However, these techniques are limited to evaluating the cellular state at a single temporal point based on snapshot analysis, thus failing to account for temporal changes and dynamic behavior of cells. Therefore, the true diversity inherent to the ever-changing HSCs remains uncertain. In this study, we comprehensively elucidate HSC diversity, including temporal changes, by integrating our recently established ex vivo HSC expansion techniques (Wilkinson et al., Nature, Becker et al., Cell Stem Cell), single-cell quantitative phase imaging (QPI), and post-transplantation dynamics analysis. For live imaging, HSCs were harvested from hepatic leukemia factor ( Hlf)-tdTomato reporter mice, which specifically marks long-term HSCs. Index sorted single Hlf-tdTomato + HSC were expanded with a single-HSC culture system and monitored by QPI fluorescence time-lapse imaging. Surprisingly, even though the cells were derived from the same HSC fraction, significant variations in mitotic rate, motility, morphological changes, and Hlf expression levels were observed at the single-cell level. Stemness did not decrease stepwise over time, but always exhibited continuous changes. Thus, analysis including the temporal dimension illuminated the infinitely branched nature of HSC diversity. Interestingly, Hlf expression level correlated with the dynamic behavior of HSCs in the expansion culture system. Hlf-high HSCs exhibited a slow division rate, small, rounded morphology and low motility, while Hlf-low HSCs actively divided and differentiated, displayed larger morphological size with low sphericity, and exhibited high motility. Furthermore, spatiotemporal dynamics analysis of Hlf-high/low HSCs after transplantation, employing the highly sensitive in vivo imaging system Akaluc bioluminescence imaging (AkaBLI), revealed that Hlf-high HSCs initiate systemic hematopoiesis slowly, while Hlf-low HSCs expanded quickly, characterized by short-term explosive hematopoiesis in the spleen. As expected, only Hlf-high HSCs had long-term bone marrow reconstitution ability. Thus, based on the expression level of Hlf, it is possible to predict the dynamic behavior of the cells in the expansion medium and in the post-transplant body. Next, we investigated whether Hlf expression level could be predicted based on the kinetic features of cells during HSC ex vivo expansion. Interestingly, multidimensional analysis based on kinetic features extracted from time-lapse imaging showed multiple clusters, each linked to Hlf expression levels. In human HSCs, the dynamic diversity of HSCs was also observed in our recently established ex vivo expansion system (Sakurai et al., Nature). Notably, the kinetic features of human HSCs differed from those of mouse HSCs. Transplantation of human HSCs exhibiting specific morphological characteristics showed long-term reconstitution ability. Thus, classification of HSCs based on kinetic features facilitated enrichment of functional HSCs. This study demonstrates the importance of characterizing cells, including the temporal dimension, and illustrates the existence of infinite dynamic individuality in mouse and human HSCs.

Demonstrating drug treatment efficacies by monitoring superoxide dynamics in human lung cancer cells with time-lapse fluorescence microscopy.

Metformin hydrochloride, an antihyperglycemic agent, and sulindac, a nonsteroidal anti-inflammatory drug, are FDA-approved drugs known to exert anticancer effects. Previous studies demonstrated sulindac and metformin's anticancer properties through mitochondrial dysfunction and inhibition of mitochondrial electron transport chain complex I and key signaling pathways. In this study, various drugs were administered to A549 lung cancer cells, and results revealed that a combination of sulindac and metformin enhanced cell death compared to the administration of the drugs separately. To measure superoxide production over time, we employed a time-lapse fluorescence imaging technique using mitochondrial-targeted hydroethidine. Fluorescence microscopy data showed the most significant increases in superoxide production in the combination treatment of metformin and sulindac. Results showed significant differences between the combined drug treatment and control groups and between the positive control and control groups. This approach can be utilized to quantify the anticancer efficacy of drugs, creating possibilities for additional therapeutic options.

N-linked glycosylation of flavivirus E protein contributes to viral particle formation.

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

Macrophage specificity: Imaging and characterization of interaction between macrophages and fluorescent organosilica nanoparticles depending on surface structure and size.

Macrophages have key functions in tissue homeostasis and also diseases. Macrophages show marked heterogeneity in response to environmental stimuli. The heterogeneity of macrophages was characterized by using morphology, surface marker, single cell sequence, and so on. We are investigating macrophage specificity using our nanomedical technology. We are developing novel materials, functional organosilica nanoparticles, and performed imaging to evaluate the diversity and specificity of mouse macrophage uptake. The organosilica nanoparticles surface-functionalized with polyethylene glycol (PEGylated) were applied to time-lapse fluorescent microscopic imaging and flow cytometry analyses. Single-cell imaging and analysis revealed that PEGylated nanoparticles revealed a stealth function against most macrophages. Notably, we found that the “PEG-resistant macrophages” that could take up PEGylated nanoparticles at the same level as bare nanoparticles. In addition, size-dependent uptake of macrophages was also observed in various tissues. The size-dependent uptake of functional organosilica nanoparticles by macrophages in the mouse various organs in vivo. These results indicated imaging and characterization of interaction between macrophages and fluorescent organosilica nanoparticles depending on surface structure and size were very useful to evaluate the heterogeneity and possible specificity of macrophage.

Establishment and characterization of the immortalized porcine lung-derived mononuclear phagocyte cell line.

Mononuclear phagocytes (MNP), including monocytes, dendritic cells (DC), and macrophages, play critical roles in innate immunity. MNP are abundant in the lungs and contribute to host defense against airborne agents and pulmonary immune homeostasis. In this study, we isolated porcine lung-derived MNP (PLuM) from primary cultures of parenchymal lung cells and then immortalized them by transferring the SV40 large T antigen gene and porcine telomerase reverse transcriptase gene using lentiviral vectors. The established cell line, immortalized PLuM (IPLuM), expressed DC/macrophage markers; i.e., CD163, CD172a, and major histocompatibility complex class II, whereas they did not express a porcine monocyte-specific marker, CD52. The expression patterns of these cell surface markers indicate that IPLuM originate from the DC/macrophage lineage rather than the monocyte lineage. The bacterial cell wall components muramyl dipeptide and lipopolysaccharide induced the production of the interleukin-1 family of pro-inflammatory cytokines in IPLuM. Phagocytotic activity was also detected by time-lapse fluorescence imaging of live cells when IPLuM were cultured in the presence of pHrodo dye-conjugated E. coli BioParticles. It is worth noting that IPLuM are susceptible to African swine fever virus infection and support the virus' efficient replication in vitro. Taken together, the IPLuM cell line may be a useful model for investigating host-agent interactions in the respiratory microenvironments of the porcine lung.

Preprint Highlight: Real-time denoising of fluorescence time-lapse imaging enables high-sensitivity observations of biological dynamics beyond the shot-noise limit.

Denoising in fluorescence imaging is a key technology to boost sensitivity and detect low signals buried in noise. While previous deep learning-based algorithms have been demonstrated to improve denoising quality, their application has been limited by the time-consuming processing step. This study demonstrates a much faster deep learning-based denoising algorithm, DeepCAD-RT, that enables real-time processing of fluorescence live cell images and can be incorporated into the data acquisition pipeline of a microscopy system. In addition to its obvious advantage in improving workflow efficiency, DeepCAD-RT could also have an impact on robotics technologies, such as automatic microinjection and cell picking, that require highly sensitive and real-time recognition of cells under a microscope.

ROR2-Vangl2 complexes direct ovarian cancer cell motility in response to Wnt5a (265)

<b>Objectives:</b> In both developmental and cancer contexts, collective migration has shown to be at least partially dependent upon asymmetric localization of core protein complexes within leader and follower cells. A critical function of non-canonical Wnt signaling in vertebrates is to regulate planar cell polarity (PCP), the polarity axis that organizes the plane of a tissue. Cells at the invasive front of cellular aggregates exhibit elevated expression of genes associated with motility pathways, including Wnt/PCP signaling. Using microfluidic devices, we have previously shown the soluble non-canonical Wnt ligand, <i>WNT5A</i>, influencing the directional motion of ovarian cancer cells via the ROR2 receptor. We sought to determine whether asymmetrical localization of ROR2 could guide <i>WNT5A-</i>directed motility. <b>Methods:</b> A DNA sequence encoding a fusion protein of ROR2 with a C-terminal Emerald (Em) fluorescent protein tag was introduced into ES2 ovarian cancer cells. Aggregated spheroids of ES2 ROR2-Em cells were plated onto a fibronectin-coated surface, and time-lapse fluorescent images were obtained of the radial migration in the presence or absence of <i>WNT5A</i>. Based on the direction of motion, cells were divided into leading or trailing half for analysis of the localization of ROR2 protein. Data were presented as mean +/- SEM. For co-immunoprecipitation experiments, ES2 cells expressing a Myctagged ROR2 DNA construct were utilized, with Myc epitope used for pulldown. <b>Results:</b> In the absence of <i>WNT5A</i>, ROR2 was uniformly distributed among the leading and lagging edge of the migrating cells (46.9% +/-2.452 ROR2 localized on the leading edge). In the presence of <i>WNT5A</i>, ROR2 was preferentially located to the leading edge of migrating cells (64.6% +/- 3.908 ROR2 localized on the leading edge). <i>VANGL2</i>, a core component in planar cell polarity, formed Wnt-induced receptor complexes with ROR2 in developing limb buds and other tumor types. <i>VANGL2</i> had been shown to reside at the protrusive edge of leader cells. To address whether <i>VANGL2</i>-ROR2 complexes contributed to the leading-edge localization of ROR2 in ovarian cancer cells, co-IP experiments were performed. Complex formation of ROR2-<i>VANGL2</i> was present in ovarian cancer cells at baseline (<i>VANGL2</i>:ROR2 IP ratio 1), yet was increased in the presence of <i>WNT5A</i> (<i>VANGL2</i>:ROR2 IP ratio 1.5). <b>Conclusions:</b> We have previously shown that <i>WNT5A</i> promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as the degree of displacement in a ROR2 dependent manner. Asymmetric localization of Wnt/PCP core component complexes within leader and follower cells is crucial for cell motility, an intrinsic metastatic property. Our studies suggest that under the influence of <i>WNT5A</i>, ROR2 localizes to the leading edge of migrating cells. Additionally, ROR2 complexes with <i>VANGL2</i> in ovarian cancer cells, and this complex is enhanced in the presence of <i>WNT5A</i>. As <i>WNT5A</i> is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the microenvironment of ovarian cancer metastasis, disruption of the ROR2-<i>VANGL2</i> complex represents an attractive target for disrupting metastatic colonization of ovarian tumors.

Classification of antimicrobial mechanism of action using dynamic bacterial morphology imaging

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Controllable Polymerization of N -Substituted β-Alanine N -Thiocarboxyanhydrides for Convenient Synthesis of Functional Poly(β-peptoid)s

Open AccessCCS ChemistryRESEARCH ARTICLE27 May 2022Controllable Polymerization of N-Substituted β-Alanine N-Thiocarboxyanhydrides for Convenient Synthesis of Functional Poly(β-peptoid)s Ximian Xiao†, Min Zhou†, Zihao Cong, Longqiang Liu, Jingcheng Zou, Zhemin Ji, Ruxin Cui, Yueming Wu, Haodong Zhang, Sheng Chen, Maoquan Li and Runhui Liu Ximian Xiao† State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 †X. Xiao and M. Zhou contributed equally to this work.Google Scholar More articles by this author , Min Zhou† State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 †X. Xiao and M. Zhou contributed equally to this work.Google Scholar More articles by this author , Zihao Cong Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Longqiang Liu Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Jingcheng Zou Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Zhemin Ji Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Ruxin Cui Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Yueming Wu Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Haodong Zhang Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Sheng Chen Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author , Maoquan Li Department of Interventional and Vascular Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072 Google Scholar More articles by this author and Runhui Liu *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 Key Laboratory for Ultrafine Materials of Ministry of Education, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, Research Center for Biomedical Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202201840 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Poly(β-peptoid) is a class of polypeptide mimics that possesses excellent biocompatibility and resistance to proteolysis. However, the synthesis of poly(β-peptoid)s with functionalities is a long-standing challenge that greatly hinders the functional study and application of poly(β-peptoid)s. We report a controllable and easy synthesis of poly(β-peptoid)s bearing diverse functionalities via the ring-opening polymerization on N-substituted β-alanine N-thiocarboxyanhydrides (β-NNTAs). The polymerization can be carried out in open vessels under mild conditions using amines as the initiators to provide poly(β-peptoid)s with targeted molecular weights, narrow dispersities, and diverse functionalities in the side chains and termini. The β-NNTAs polymerization is even compatible with initiators bearing unprotected hydroxyl groups. The amphiphilic/cationic poly(β-peptoid)s exhibit a broad spectrum and potent antibacterial activities against multidrug-resistant bacteria. In addition, the highly favored stability of β-NNTA monomers for purification and storage highlights the advantages of this β-NNTA polymerization strategy for poly(β-peptoid)s synthesis, functional study, and application as a synthetic mimic of polypeptides. Download figure Download PowerPoint Introduction Poly(β-peptoid)s, as a class of polypeptide mimics, have a polyamide backbone similar to polypeptides but with an extra methylene group and with the proton of nitrogen–hydrogen bond in the backbone substituted by an alkyl group.1–4 Poly(β-peptoid)s have superior biocompatibility and excellent stability against proteolysis and therefore demonstrate promising biological applications in biomimetic scaffolds,3 antifouling properties,5 and antibacterials.6 However, the lack of an effective synthetic strategy is the long-standing challenge that greatly hinders the advance of this field and the application of poly(β-peptoid)s. Conventional solid-phase synthesis is applied to prepare poly(β-peptoid)s using either direct amide bond formation or via aza-Michael addition.7,8 However, no poly(β-peptoid)s with over 10 residues can be obtained using the solid-phase synthesis due to the low efficiency of this chemistry. Compared with the preparation of polypeptides and poly(α-peptoid)s, it is hard to obtain structurally diverse and functional poly(β-peptoid)s. Current solid-phase synthesis of poly(β-peptoid)s is also time-consuming, expensive, and difficult in scale up.9 To overcome the shortcomings of solid-phase synthesis in preparing poly(β-peptoid)s, polymerization strategies have been explored. Jia et al. reported a successful synthesis of poly(β-peptoid)s from the copolymerization of N-alkylaziridines and carbon monoxide using cobalt catalysts (Figure 1a).1,10 Nevertheless, this polymerization relies on high-pressure reaction conditions and a metal catalyst. Since 1954, to realize a mild synthesis of poly(β-peptoid)s, amine-initiated polymerization on N-substituted β-alanine N-carboxyanhydrides (β-NNCAs) has been explored (Figure 1b).2,11,12 However, this strategy suffers from the instability of β-NNCA monomers.12 In addition, the above two polymerization strategies for poly(β-peptoid)s synthesis are both highly sensitive to moisture and have structural diversity limited to N-alkyl and/or N-aryl substitution, which has been a serious obstacle to the synthesis, functional study, and application of poly(β-peptoid)s. Therefore, we need to develop a convenient, mild, and open vessel synthesis of poly(β-peptoid)s. Meanwhile, it is also vital to explore structurally diverse poly(β-peptoid)s for their functions and applications. Figure 1 | Polymerization strategy for the synthesis of poly(β-peptoid)s. Download figure Download PowerPoint Herein, we report the synthesis of poly(β-peptoid)s from a controllable ring-opening polymerization on N-substituted β-alanine N-thiocarboxyanhydrides (β-NNTAs) (Figure 1c). This controllable and living polymerization on β-NNTAs can be achieved using variable amines as the initiator to obtain poly(β-peptoid)s with predictable molecular weight (Mn) controllable degree of polymerization (DP) and low dispersity (Đ) without using any catalyst. This polymerization can be operated conveniently in an open vessel. Moreover, β-NNTA monomers can be easily obtained with a variety of N-substitutions, which enables a breakthrough in the structural diversity of resulting poly(β-peptoid)s. In addition, a variety of terminal functional groups can be introduced into poly(β-peptoid)s via the amine initiators. This strategy for poly(β-peptoid)s synthesis from controllable and living polymerization on β-NNTAs addresses the long-lasting challenges in this field and provides a convenient alternative to facilitate the synthesis, functional study, and application of poly(β-peptoid)s. Experimental Methods All reagents and solvents were purchased from Adamas Chemical Reagent Co., Ltd. (Shanghai, China) and used as received unless otherwise specified. Petroleum ether (PE), ethyl acetate (EtOAc), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), toluene (Tol), acetonitrile (MeCN), dioxane, dimethyl sulfoxide (DMSO), and 1-methyl-2-pyrrolidinone (NMP) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. and used as received without purification. Synthesized intermediates were purified using a SepaBean machine equipped with Sepaflash columns produced by Santai Technologies (Changzhou, China) Co. Ltd. Nuclear magnetic resonance (NMR) spectra were collected on a Bruker spectrometer at 400 MHz or 600 MHz, using CDCl3 or CF3COOD as solvents. Chemical shifts are referenced to residual protons in the deuterated NMR solvents. High-resolution electrospray ionization time-of-flight mass spectrometry (HRESI-TOF-MS) was collected on a Waters Xevo G2 TOF mass spectrometer (Waters Technologies Shanghai Ltd) and high-resolution electron ionization time-of-flight mass spectrometry (HREI-TOF-MS) was collected on a Waters GCT. High-performance liquid chromatography (HPLC) analysis was carried out on a Shimadzu LC-20AR HPLC System (Shimadzu (Shanghai) Global Laboratory Consumables Co., Ltd.) equipped with a Gemini 5 μm NX-C18 column. Gel permeation chromatography (GPC) was performed on a Waters GPC instrument equipped with a Brookhaven BI-MwA multiangle light-scattering detector (BI-MwA, Brookhaven Instruments Shanghai Representative Office) and a refractive index detector (Waters 2414) using DMF, supplemented with 0.01 M LiBr, as the mobile phase at a flow rate of 1 mL/min at 50 °C. The GPC were equipped by a Tosoh TSKgel Alpha-2500 column (particle size 7 μm), a Tosoh TSKgel Alpha-3000 column (particle size 7 μm), and a Tosoh TSKgel Alpha-4000 column (particle size 10 μm, 300 × 7.8 mm) linked in series. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) experiments were conducted on an AB Sciex 4800 Plus MALDI TOF/TOF analyzer (Shanghai AB Sciex Analytical Instrument Trading Co., Ltd.) using a 355 nm laser, a Shimadzu MALDI-7090TM TOF-TOF mass spectrometer (Shimadzu (Shanghai) Global Laboratory Consumables Co., Ltd.) with 2,5-dihydroxybenzoic acid (DHB) as the matrix. Dynamic light scattering (DLS) measurements were used to measure hydrodynamic diameters of assembled polymers using a Zetasizer Nano-ZS instrument, Model ZEN3600 (Malvern Instruments Ltd., Malvern, United Kingdom). Transmission electron microscopy (TEM) measurements were conducted using a JEOL JEM-1400 TEM (JEOL (Beijing) Co., Ltd. Shanghai Branch) at an acceleration voltage of 100 kV. Time-lapse fluorescent confocal imaging was performed using a Leica TCS SP8 system (Leica Microsystems) equipped with LAS X v3.5.2. software (Leica Microsystems) for acquisition control. Fluorescence spectra of the samples were performed on a Varian Cary Eclipse fluorescence spectrophotometer (Varian China Ltd., Shanghai Office). Results and Discussion Using pFBn β-NNTA ( 1a) as a model, we conducted the ring-opening polymerization on β-NNTA using 4-tert-butylbenzylamine (tBuBnNH2) as the initiator in variable solvents including DMF, THF, Tol, dioxane, MeCN, DMSO, and NMP ( Supporting Information Scheme S13, Figure S1, Table S1). Poly(N-pFBn-β-ala) was successfully obtained in polymerization using most common solvents as a single peak of GPC trace with narrow dispersity (Đ = 1.04–1.11), except that a bimodal peak of GPC trace was observed for polymerization using DMSO and NMP as the solvent. Among all solvents that are compatible with β-NNTA polymerization, DMF afforded the resulting poly(β-peptoid) with a polymer length (obtained DP = 21) almost identical to the expectation (calculated DP = 20). Considering that DMF normally provides excellent solubility and is a widely used solvent for polypeptide and polypeptoid synthesis, we chose DMF as the solvent for continuous β-NNTA polymerization studies. To examine if the polymer length of amine-initiated β-NNTA polymerization is controllable, we tried the polymerization on pFBn β-NNTA ( 1a) in DMF using variable [M]0/[I]0 ratios. The results showed that poly(β-peptoid)s were obtained with an incrementally increased chain length from 22 mer (Mn = 4.1 kDa) to 150 mer (Mn = 27.1 kDa) with narrow dispersities (Đ = 1.18–1.20) and DP almost identical to the prediction (Table 1, entries 1–5, Figure 2a). These results demonstrate the excellent controllability of the β-NNTA polymerization. Kinetics study showed that the polymerization had an evolution of Mn that showed in linear correlation with monomer conversion and kept narrow dispersity (Đ = 1.09–1.18) throughout the whole process (Figure 2b). It is worth mentioning that the polymerization showed first-order kinetic characteristics (kp[I] = 0.018 h−1) and over 90% conversion of β-NNTA monomer within 14 h (Figure 2c). The polymerization on pFBn β-NNTA in DMF was completed within 20 h. Table 1 | Poly(β-peptoid)s Synthesized from β-NNTAs Polymerizationa Entry M M0/I Mn, th (kDa) Mn (kDa)b DPb Ðb 1 1a 20 3.7 4.1 22 1.19 2 1a 40 7.3 7.3 40 1.20 3 1a 80 14.6 14.6 80 1.19 4 1a 100 18.1 17.9 99 1.18 5 1a 150 27.0 27.1 150 1.20 6 1b 20 3.4 3.2c 19c 1.12d 7 1c 20 4.3 4.6 21 1.13 8 1d 40 7.4 7.2 39 1.18 9 1e 20 2.4 2.1c 17c 1.15d 10 1f 20 2.7 2.4 18 1.17 11 1g 20 2.7 3.6c 27c 1.08d 12 1h 20 4.8 3.3 14 1.09 13 1i 20 5.1 4.0 16 1.13 14 1a-r- 1d 40/40 14.6 13.9 76 1.17 15 1a1st 20 3.7 3.7 20 1.16 1d2nd 20 7.4 7.3 40 1.15 aAll polymerizations were conducted using tBuBnNH2 as initiator in a sealed vessel at 60 °C. [M]0 at 2 M for entry 5, [M]0 at 1 M for other entries. bMn, DP, and Đ were determined by GPC using DMF as the mobile phase at a flow rate of 1 mL/min. cMn and DP were determined by NMR. dĐ was determined by MALDI-TOF-MS. Mn,th is the theoretical number average molecular weight; Mn,obtained is the obtained number average molecular weight; DP is the obtained degree of polymerization; Đ means the dispersity. Figure 2 | (a) Plots of Mn and Đ as a function of the [M]0/[I]0 ratio for pFBn β-NNTA. Inset: Overlay of GPC profiles at different [M]0/[I]0 ratios corresponding to entries 1–5 in Table 1. (b) Profiles of Mn and Đ as a function of pFBn β-NNTA conversions at the [M]0/[I]0 ratio of 20/1. Inset: Overlay of GPC profiles at different monomer conversions. (c) Kinetic profiles of conversion and ln([M]0/[M]) vs time at the [M]0/[I]0 ratio for pFBn β-NNTA of 20/1 using tBuBnNH2 as the initiator. (d) GPC traces of the block copolymer corresponding to entry 15 in Table 1. (e) The ring-closure synthesis for β-NNTAs. Reaction condition: 1.0 equiv. PBr3, anhydrous CH2Cl2, 0 °C to r.t., 4 h. (f) The purification of Bu β-NNTA via silica gel chromatography (9:1 PE:EtOAc). (g) Stability analysis of β-NNTA and β-NNCA at r.t. (h) Open-vessel polymerization of ThEt β-NNTA at 2 g scale at 60 °C in DMF, [M]0 = 1 M. Mn, DP and Đ were determined by GPC. Download figure Download PowerPoint The side-chain functionality or structural diversity of peptide mimics plays an essential role in defining their activity, function, and application.13–17 However, for poly(β-peptoid)s, current synthetic strategies using either the copolymerization of N-alkylaziridines and carbon monoxide or the ring opening polymerization of β-NNCAs can only afford poly(β-peptoid)s with N-alkyl or N-aryl functional groups.1,2 This challenge encouraged us to explore the synthesis of functional poly(β-peptoid)s from the polymerization on β-NNTAs. Structurally diversified β-NNTAs were easily synthesized from primary amines (Figure 1c, β-NNTA 1a– 1i, Supporting Information Schemes S1–S10 and Figures S32–S57). In addition, tBuBnNH2 successfully initiated the polymerization of all those β-NNTAs in our study to afford corresponding poly(β-peptoid)s in narrow dispersities (Đ = 1.09–1.18) and with diversified structures or side-chain functional groups including aromatic groups (4-fluorobenzyl in pFBn β-NNTA, benzyl in Bn β-NNTA, (4-chlorophenyl)ethyl in pCPE β-NNTA), heterocycles in ThEt β-NNTA, alkyl groups (cyclopropyl in cPr β-NNTA, isobutyl in iBu β-NNTA, and n-butyl in Bu β-NNTA), carboxylates in Cbz-Et β-NNTA, and amines in Cbz-AE β-NNTA (Table 1, entries 1–13, Figure 2a, and Supporting Information Figures S2–S9). The β-NNTA polymerization was also feasible for copolymerization. A random copolymerization on the mixture of pFBn β-NNTA and ThEt β-NNTA afforded poly[(N-pFBn-β-ala)-r-(N-ThEt-β-ala)] with the expected polymer length (DP = 76) and a narrow dispersity (Đ = 1.17) (Table 1, entry 14, Supporting Information Scheme S14 and Figure S10). Sequential addition of pFBn β-NNTA and ThEt β-NNTA in one-pot synthesis gave the first block of poly(N-pFBn-β-ala) at 20 mer with a narrow dispersity (Đ = 1.16) and the expected final block copolymer poly(N-pFBn-β-ala)-b-poly(N-ThEt-β-ala) at 40 mer with a low dispersity (Đ = 1.15) (Table 1, entry 15, Figure 2d, and Supporting Information Scheme S15). All these results demonstrate that the amine-initiated β-NNTA polymerization has excellent controllability to prepare poly(β-peptoid)s with diversified structures and side-chain functionalities. The stability of β-NNTA is vital to its purification and storage, ultimately contributing to structural and functional diversity of poly(β-peptoid)s. We found that β-NNTA displayed stability superior to β-NNCA with the same side chain. It is worth mentioning that we can obtain all β-NNTAs with high yield (81−93%) during the ring-closing process via silica gel chromatography (Figure 2e). Purification of β-NNTA via silica gel chromatography makes it possible to easily obtain pure oily β-NNTA monomers (representative Bu β-NNTA in Figure 2f), whereas β-NNCA is less stable and will decompose on silica gel, making the purification of oily β-NNCA monomers a headache ( Supporting Information Scheme S11 and Figures S58–S60). In addition, during storage, the purity of β-NNTA such as pFBn β-NNTA was maintained above 99% over 27 days at room temperature and showed no significant decomposition even after half a year at −20 °C (Figure 2g, Supporting Information Figure S11), whereas the purity of the corresponding β-NNCA sharply decreased to 23% after 27 days (Figure 2g). The open-vessel polymerizations have been reported in the synthesis of polypeptides and polypeptoids.18–27 The excellent stability of β-NNTA encouraged us to explore its polymerization in an open flask outside of a glove box. A gram-scale β-NNTA polymerization was achieved successfully in an open flask to give the resulting poly(N-ThEt-β-ala)20 in 90.1% yield (Figure 2h). The living and controllable polymerization of β-NNTAs encouraged us to explore the reaction mechanism. We proposed that the polymerization is initiated by nucleophilic addition of primary amine at the C1 carbonyl in a β-NNTA ring, followed by the ring opening of β-NNTA and release of the small molecule carbonyl sulfide (COS) to give an intermediate 1 that has an N-terminal secondary amine as the reactive center to attack another β-NNTA for chain propagation (Figure 3a).24,26,28,29 To provide mechanistic insights into chain initiation and propagation, Fourier transform infrared (FT-IR) characterization on a 1∶1 molar ratio mixture of tBuBnNH2 and pFBn β-NNTA was conducted (Figure 3b). Compared with pFBn β-NNTA, an above 1∶1 mixture had a shifted signal of the C1 carbonyl group from 1715.9 to 1642.1 cm−1 (C1′ in the mixture). The disappearance of C2 carbonyl signal at 1648.8 cm−1 indicated the ring opening of β-NNTA and subsequent release of COS. In addition, proton NMR characterization of an immediately prepared mixture of pFBn β-NNTA and tBuBnNH2 solution in CDCl3 at variable ratios showed a reduction of Ha and Hb peaks in the β-NNTA ring and the emergence of upfield-shifted Ha′ and Hb′ peaks in the β-NNTA ring opening product simultaneously, which indicated a quick nucleophilic addition of tBuBnNH2 to the C1 carbonyl in the β-NNTA ring at the initiation step (Figure 3c). Figure 3 | (a) Proposed mechanism of primary amine-initiated β-NNTA polymerization. (b) FT-IR spectra of β-NNTA, tBuBnNH2 and a 1∶1 β-NNTA/tBuBnNH2 mixture (DMF, room temperature, [M]0=0.5 M). (c) Instantaneous 1H NMR titration spectra on a mixture of β-NNTA/tBuBnNH2 in variable ratio (CDCl3, 25°C. [M]0=0.2 M). (d) HRESI-TOF-MS analysis on the 1∶1 β-NNTA/tBuBnNH2 mixture (DMF, room temperature, [M]0=0.5 M). (e) MALDI-TOF-MS of poly(N-pFBn-β-ala)20 (entry 1 in Table 1) with the corresponding chemical structures. Download figure Download PowerPoint HRESI-TOF-MS characterization on the above 1∶1 mixture showed a clear spectrum and high intensity fragment at m/z 343.2187 for intermediate 1 (Figure 3d), consistent with the result in the above NMR analysis and confirming nucleophilic addition of tBuBnNH2 to the β-NNTA ring during initiation. This was further confirmed by examining the final poly(β-peptoid)s (poly(N-pFBn-β-ala)20) using MALDI-TOF-MS characterization. The 179 Da difference between peaks reflected successful incorporation of the β-peptoid residues. Specific mass corresponding to each peak revealed the incorporation of a C-terminal tBuBnNH group via nucleophilic addition during initiation (Figures 3a and 3e). These results echoed the controllable β-NNTA polymerization and living polymerization with a normal amine mechanism. The terminal functionalization is important for the structural diversity and function of peptide mimics, for instance, in bioactive molecule conjugation and fluorescence labelling.30–39 The aforementioned open-vessel polymerization encouraged us to further explore C-terminal functionalization in an open vessel ( Supporting Information Scheme S16). Using primary amines as the initiator for the ring-opening polymerization on β-NNTAs allows the easy incorporation of diverse functionalities into the C-terminal of poly(β-peptoid) chains including alkenyl, alkynyl, adamantane, bromophenyl, azide, protected thiol, protected amine, methoxy, polyethylene glycol (Figure 4a, 2a− 2j), unprotected base, unprotected hydroxyl groups, aggregation-induced emission luminogen (AIEgen), and aggregation-caused quenching (ACQ) fluorophore (Figures 4b and 4c, 2k− 2q). In addition to terminal functionalization, all poly(β-peptoid)s were successfully synthesized with excellent control of chain length and narrow dispersities (Đ = 1.06−1.08) ( Supporting Information Figures S12−S29). Figure 4 | Primary amine-initiated β-NNTA polymerization in an open vessel. (a) Different functional primary amines as C-terminal groups. (b) Representative primary amines with nucleophilic groups. (c) Functional demonstration of C-terminal modified poly(β-peptoid)s. Download figure Download PowerPoint What is more, the polymerization on β-NNTAs can be initiated by primary amines that have an unprotected base (pyridine, tertiary amine) and hydroxyl group (phenol, alcohol) (Figure 4b, 2k– 2n). The tolerance of unprotected reactive groups of β-NNTA polymerization facilitated the synthesis of poly(β-peptoid)s with function groups without tedious protection and deprotection steps. This encouraged us to explore a more challenging β-NNTA polymerization using unprotected 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin (NH2-β-CD) as the initiator. Surprisingly, the polymerization also tolerated multiple hydroxyl groups to give the expected poly(β-peptoid)s smoothly with a narrow dispersity (Đ = 1.15, GPC analysis) and a terminal β-CD moiety in the polymer chain as characterized by MALDI-TOF MS (Figure 4c, 2o, Supporting Information Figure S27). The β-CD grafted poly(β-peptoid)s spontaneously self-assembled in the mixing solvent of water/DMSO, indicating the potential application of this polymerization in drug delivery (Figure 4c, 2o).25,40–46 This mild β-NNTA polymerization also enabled us to synthesize poly(β-peptoid)s with terminal fluorescence groups such as the AIEgen tetraphenylethylene ( 2p-poly(N-pFBn-β-ala)20) ( Supporting Information Scheme S12 and Figures S61 and S62) and the ACQ fluorophore morpholino-naphthalimide ( 2q-poly(N-pFBn-β-ala)20), with the expected polymer length and low dispersity (Figure 4c, 2p and 2q). 2p-poly(N-pFBn-β-ala)20 and 2q-poly(N-pFBn-β-ala)20 displayed AIE and ACQ characteristics, respectively. They showed the strongest photoluminescence (PL) intensity at 90% and 0% water content, respectively, which indicates a potential application of this β-NNTA polymerization and resulting poly(β-peptoid)s for imaging.30,47–51 Poly(β-peptoid)s have backbone structures similar to polypeptides, favorable biocompatibility, and excellent stability against proteolysis, which strongly suggests the possible use of poly(β-peptoid)s as a synthetic mimic of polypeptides for a large variety of applications. However, current functional study and application of poly(β-peptoid)s are very limited due to the obstacle of poly(β-peptoid) synthesis.28,52 To the best of our knowledge, no antibacterial application of poly(β-peptoid)s has been reported, likely due to the inaccessibility to poly(β-peptoid)s via polymerization.6,9,52 The mild and controllable synthesis of poly(β-peptoid)s from β-NNTA polymerization enables us to prepare structurally diverse poly(β-peptoid)s for functional studies including antibacterials. Using Cbz-AE β-NNTA and pFBn β-NNTA as models for proof-of-concept demonstrations, we synthesized amphiphilic poly(β-peptoid)s (poly[(N-Cbz-AE-β-ala)-r-(N-pFBn-β-ala)]20) that were composed of both the cationic subunit (aminoethyl functionality) and the hydrophobic subunit (4-fluorobenzyl functionality) with an incrementally increased ratio of the cationic subunit as synthetic mimics of host defense peptides (Figures 5a and 5b).13,23,53–62 These amphiphilic poly(β-peptoid)s were obtained at about 20 mer at low dispersities (Đ = 1.12−1.13) (Figures 5a and 5b, GPC traces in Supporting Information Figure S30). Figure 5 | Open-vessel polymerization for synthesis of poly(β-peptoid)s for antibacterial activity studies. (a) A library of amphiphilic poly(β-peptoid)s. (b) Characterizations of poly(β-peptoid)s. (c) The MIC values of the amphiphilic poly(β-peptoid)s against 5 strains of drug-resistant bacteria. (d) GPC characterization of side-chain protected poly[(N-Cbz-AE-β-al

NCAD and ITGB1 Coordinately Regulate Growth Plate Cartilage Architecture

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives long bone growth. A notable feature of growth plate cartilage is the progression of progenitor resting chondrocytes into clonal columns of transit‐amplifying proliferative chondrocytes. Following division of proliferative chondrocytes, daughter cell pairs undergo a unique 90‐degree rearrangement to form columns. Our previous studies using a murine fluorescent membrane reporter system and time‐lapse imaging along with chemical inhibition of cell adhesion suggested a primary requirement for cadherin adhesion at the daughter cell interface in column formation. Studies designed to test the force generating mechanism at the cadherin dependent daughter cell interface produced a paradoxical result with activated myosin motor proteins enriched at the cell‐matrix interface, an observation that suggests integrin adhesion molecules might drive cell rearrangement. The purpose of the current work was to determine the relative contributions of cadherin and integrin mediated cell adhesion in chondrocyte column formation. As a first step, we used live cell imaging to compare the daughter cell rearrangement phenotype in growth plate explants from embryos containing a cartilage‐specific deletion of N‐cadherin (NCAD) or integrin β1 (ITGB1). Deletion of either gene produces similar disruption of column formation, even though the mutant phenotypes show differences in cell shape, rearrangement dynamics, and kinetics of daughter cell separation following rearrangement. The apparent requirement for both NCAD and ITGB1 suggested potential reciprocal regulatory interaction between the two adhesion surfaces. To test this hypothesis, we analyzed phenotypes of an allelic series. We found that chondrocytes mutant for ITGB1 show defects in cell rearrangement and column formation (n=37 embryos) regardless of the NCAD genotype as expected from single mutant studies. Surprisingly, chondrocytes mutant for NCAD and heterozygous for ITGB1 are phenotypically wildtype with organized columns and normal rearrangement (n=13 embryos). These results support a model for which ITGB1, and not NCAD, is the driver of chondrocyte rearrangement and suggest that failure to rearrange in NCAD mutants results from increased ITGB1 function. Thus, rearrangement requires a narrow range of cell‐matrix adhesion strength and NCAD potentially acts as a brake by restricting ITGB1 activity in non‐rearranging resting chondrocytes. Current studies aim to confirm these genetic findings using gene expression analysis and an innovative quantitative adhesion assay as well as identify upstream regulators that control strength of antagonism between the cell adhesion interfaces.

Monitoring Mitochondrial Membrane Potential in Live Cells Using Time-Lapse Fluorescence Imaging.

The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III, and IV) is an essential component in the process of energy generation during oxidative phosphorylation. Tetramethylrhodamine, methyl ester, perchlorate (TMRM) is one of the most commonly used fluorescent reporters of ΔΨm. TMRM is routinely employed in a steady state for the measurement of membrane potential. However, it can also be utilized with time-lapse fluorescence imaging to effectively monitor the changes in membrane potential in response to a given stimulus by analyzing the change in distribution of the dye with time.

Fluorescence Time-lapse Imaging of Entosis Using Tetramethylrhodamine Methyl Ester Staining.

Entosis is a process where a living cell launches an invasion into another living cell's cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining. This protocol was validated in: J Cell Biol (2021), DOI: 10.1083/jcb.202010030.

Zinc is an intracellular signal during sperm activation in Caenorhabditis elegans.

Sperm activation is a rapid and dramatic cell differentiation event that does not involve changes in transcription, and the signaling cascades that mediate this process have not been fully defined. zipt-7.1 encodes a zinc transporter, and zipt-7.1(lf) mutants display sperm-activation defects, leading to the hypothesis that zinc signaling mediates sperm activation in Caenorhabditis elegans. Here, we describe the development of a method for dynamic imaging of labile zinc during sperm activation using the zinc-specific fluorescence probe FluoZin-3 AM and time-lapse confocal imaging. Two phases of dynamic changes in labile zinc levels were observed during sperm activation. Forced zinc entry using the zinc ionophore pyrithione activated sperm in vitro, and it suppressed the defects of zipt-7.1(lf) mutants, indicating that high levels of cytosolic zinc are sufficient for sperm activation. We compared activation by zinc pyrithione to activation by extracellular zinc, the Na+/H+ antiporter monensin and the protease cocktail pronase in multiple mutant backgrounds. These results indicate that the protease pathway does not require zinc signaling, suggesting that zinc signaling is sufficient to activate sperm but is not always necessary.

Streptomyces venezuelae NRRL B-65442: genome sequence of a model strain used to study morphological differentiation in filamentous actinobacteria.

For over a decade, Streptomyces venezuelae has been used to study the molecular mechanisms that control morphological development in streptomycetes and is now a well-established model strain. Its rapid growth and ability to sporulate in a near-synchronised manner in liquid culture, unusual among streptomycetes, greatly facilitates the application of modern molecular techniques such as ChIP-seq and RNA-seq, as well as time-lapse fluorescence imaging of the complete Streptomyces life cycle. Here we describe a high-quality genome sequence of our isolate of the strain (Northern Regional Research Laboratory [NRRL] B-65442) consisting of an 8.2 Mb chromosome and a 158 kb plasmid, pSVJI1, which had not been reported previously. Surprisingly, while NRRL B-65442 yields green spores on MYM agar, the American Type Culture Collection (ATCC) type strain 10712 (from which NRRL B-65442 was derived) produces grey spores. While comparison of the genome sequences of the two isolates revealed almost total identity, it did reveal a single nucleotide substitution in a gene, vnz_33525, involved in spore pigment biosynthesis. Replacement of the vnz_33525 allele of ATCC 10712 with that of NRRL B-65442 resulted in green spores, explaining the discrepancy in spore pigmentation. We also applied CRISPR-Cas9 to delete the essential parB of pSVJI1 to cure the plasmid from the strain without obvious phenotypic consequences.

A random-sampling approach to track cell divisions in time-lapse fluorescence microscopy

BackgroundParticle-tracking in 3D is an indispensable computational tool to extract critical information on dynamical processes from raw time-lapse imaging. This is particularly true with in vivo time-lapse fluorescence imaging in cell and developmental biology, where complex dynamics are observed at high temporal resolution. Common tracking algorithms used with time-lapse data in fluorescence microscopy typically assume a continuous signal where background, recognisable keypoints and independently moving objects of interest are permanently visible. Under these conditions, simple registration and identity management algorithms can track the objects of interest over time. In contrast, here we consider the case of transient signals and objects whose movements are constrained within a tissue, where standard algorithms fail to provide robust tracking.ResultsTo optimize 3D tracking in these conditions, we propose the merging of registration and tracking tasks into a registration algorithm that uses random sampling to solve the identity management problem. We describe the design and application of such an algorithm, illustrated in the domain of plant biology, and make it available as an open-source software implementation. The algorithm is tested on mitotic events in 4D data-sets obtained with light-sheet fluorescence microscopy on growing Arabidopsis thaliana roots expressing CYCB::GFP. We validate the method by comparing the algorithm performance against both surrogate data and manual tracking.ConclusionThis method fills a gap in existing tracking techniques, following mitotic events in challenging data-sets using transient fluorescent markers in unregistered images.