Cell Structure and Function

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

Phospholipase D (PLD) and phosphatidic acid (PA) play a spatio-temporal role in regulating diverse cellular activities. Although current methodologies enable optical control of the subcellular localization of PLD and by which influence local PLD enzyme activity, the overexpression of PLD elevates the basal PLD enzyme activity and further leads to increased PA levels in cells. In this study, we employed a split protein reassembly strategy and optogenetic techniques to modify superPLD (developed by Jeremy Baskin group). We splited this variants into two HKD domains and fused these domains with optogenetic and chemogenetic elements and by which we achieved control of the two HKD interaction and then restored the PLD enzymatic activity.

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution. Take AwayO_LIspPKA-KTR1.0 allows visualization of PKA activity at the single-cell level C_LIO_LILive-cell imaging reveals the transient decrease in PKA activity after M-phase C_LIO_LIOptogenetics allows simultaneous visualization and manipulation of PKA activity C_LI

Multi-imaging analysis has become an indispensable technique to visualize multiple target proteins and intracellular components simultaneously. While current multi-imaging analysis relies on the differences in emission spectra of fluorescent molecules, the use of fluorescence lifetime imaging microscopy (FLIM), which exploits the differences in fluorescence lifetimes of fluorescent proteins, in multi-imaging analysis is quite limited. In this study, we successfully discriminated fluorescent proteins with similar colors but different fluorescence lifetimes in vitro and in planta. We found that four fluorescent proteins with similar emission spectra could be distinguished by FLIM. In addition, we found that FLIM could clearly separate fluorescent proteins if they differ by at least 0.2 ns. In a proof-of-concept experiments for plant live imaging, we transiently expressed fluorescent proteins with different subcellular localization tags in Physcomitrium patens by particle bombardment. Each fluorescent protein exhibited its fluorescence lifetime at the subcellular localization corresponding to the localization tag in P. patens with little or no effect of chlorophyll autofluorescence. Our results demonstrate the effectiveness of FLIM in revealing the spatiotemporal dynamics of a large number of fluorescent proteins in living plant cells.

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by macropinosome formation. A certain number of mammalian mechanosensors are sensitive to membrane deformation and tension. However, it remains unclear whether macropinocytosis is regulated by mechanosensors. Focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). Although studies with Piezo1 knockout cells suggest that Piezo1 itself is not physiologically indispensable for macropinocytosis regulation, Yoda1 inhibited ruffle formation depending on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. Moreover, Yoda1 impaired cancer cell proliferation, suggesting the impact of macropinocytosis inhibition. We propose the potential for cancer therapy by macropinocytosis inhibition through the regulation of a mechanosensitive channel activity.

Chloroplasts are essential organelles in plants that are involved in plant development and photosynthesis. Accurate quantification of chloroplast numbers is important for understanding the status and type of plant cells, as well as assessing photosynthetic potential and efficiency. Traditional methods of counting chloroplasts using microscopy are time-consuming and face challenges such as the possibility of missing out-of-focus samples or double counting when adjusting the focal position. Here, we developed an innovative approach called Detecting- and-Counting-chloroplasts (D&Cchl) for automated detection and counting of chloroplasts. This approach utilizes a deep-learning-based object detection algorithm called You-Only-Look-Once (YOLO), along with the Intersection Over Union (IOU) strategy. The application of D&Cchl has shown excellent performance in accurately identifying and quantifying chloroplasts. This holds true when applied to both a single image and a three-dimensional (3D) structure composed of a series of images. Furthermore, by integrating Cellpose, a cell-segmentation tool, we were able to successfully perform single-cell 3D chloroplast counting. Compared to manual counting methods, this approach improved the accuracy of detection and counting to over 95%. Together, our work not only provides an efficient and reliable tool for accurately analyzing the status of chloroplasts, enhancing our understanding of plant photosynthetic cells and growth characteristics, but also makes a significant contribution to the convergence of botany and deep learning. One-sentence summaryThis deep learning-based approach enables the accurate complete detection and counting of chloroplasts in 3D single cells using microscopic image stacks, and showcases a successful example of utilizing deep learning methods to analyze subcellular spatial information in plant cells. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/) is: Zhao Dong (dongzhao@hebeu.edu.cn), Shaokai Yang, (shaokai1@ualberta.ca), Ningjing Liu (liuningjing1@yeah.net), and Qiong Zhao (qzhao@bio.ecnu.edu.cn).

To understand molecular mechanism of ciliary beating motion, knowledge of location, interaction and dynamics of >400 component proteins are indispensable. While recent progress of structural biology revealed conformation and localization of >100 proteins, we still need to investigate their networking, art of their interaction and assembly mechanism. We applied CRISPR/CAS9 genome editing technique to the green algae Chlamydomonas to engineer a deletion mutant of a ciliary component, FAP263, located at the distal protrusion, and examined it structurally by cryo-electron tomography (cryo-ET) and mass spectrometry (MS). Cryo-ET and atomic model fitting demonstrated that the FAP263 deletion mutant lacks additional components, FAP78, and FAP184. Unassigned density near FAP263 in the cryo-ET map of WT cilia is likely FAP151, as suggested by cross-linking mass spectrometry. Based on the structure, we modeled how these four proteins might form a complex. Furthermore, it was shown that dynein f phosphorylation is inhibited in the FAP263 mutant, indicating an important role of this protein complex for dynein f phosphorylation. Our study demonstrates a novel approach to investigate protein networking inside cilia.

Microridges are laterally elongated membrane protrusions from the apical surface of epithelial cells. Microridges are arranged in striking maze-like patterns. They are found on various mucosal epithelia in many animals, including the skin of zebrafish, where they are required to maintain mucus on the skin surface. Recent studies have revealed molecular mechanisms of how microridiges formation involving actin and actin-regulatory proteins. However, the molecular mechanism that deforms epithelial membranes to create microridge protrusions remain unknown. We have found that one of the I-BAR domain proteins, baiap2l1a, which is known to regulate membrane curvature, is required for microridge morphogenesis. CRISPR/Cas9 knockdown showed that baiap2l1a mutant zebrafish had defects in microridge morphogenesis. Baiap2l1a mutant zebrafish had shorter and wider microridges than WT microridges. Baiap2l1a localized to microridges, and its localization proceeded microridge actin formation. Furthermore, the baiap2l1a I-BAR domain, which binds and curves membranes, was sufficient to localize to microridges in zebrafish skin cells. Structure/function experiments revealed that the I-BAR domain alone could partially rescued microridge length in baiap2l1a mutants. A 39 amino acid deletion in the I-BAR domain, which caused the loss of one -helix according to AlphaFold2 simulations, is sufficient to impair microridge localization and failed to rescue microridge elongation in baiap2l1a mutants. These results suggest that the I-BAR domain is required for baiap2l1a microridge localization and function. Eps8like1a, a member of the Eps8 family proteins known as an actin capping and bundling protein genetically interacted with baiap2l1a in microridge elongation. Together, we found that the membrane curvature protein baiap2l1a plays an important role in generating microridges in zebrafish epithelia.

Transgene expression in eHAP cells, a haploid cell line popularly used to generate gene knockouts, is difficult owing to its low transfection efficiency and propensity for silencing integrated transgenes. To simplify transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells. We further demonstrate that transgenic haploid eHAP cells can be fused to form transgenic heterozygous diploid cells. This method creates diploid cells carrying the transgenic material of the haploid progenitors and could also be used to create heterozygous cells of defined genotypes. These tools expand the repertoire of experiments that can be performed in eHAP cells and other cultured cells.

For organisms that respond to environmental stimuli using taxes, reversal of the tactic sign should be tightly regulated for survival. The biciliate green alga Chlamydomonas reinhardtii is an excellent model for studying reversal between positive and negative phototaxis. C. reinhardtii cells change swimming direction by modulating the balance of beating forces between their two cilia after photoreception at the eyespot; however, it remains unknown how they reverse phototactic sign. In this study, we observed cells undergoing phototactic turns with a high-speed camera and found that two key factors determine the phototactic sign: which of the two cilia beats more strongly for phototactic turning and when the strong beating starts. The timing of the strong ciliary beating is suggested to be regulated by ROS-regulated switching between the light-on and light-off responses at the eyespot, which leads to the switching between positive and negative phototaxis. This idea is supported by a mathematical model that introduces the timing of the strong ciliary beating after photoreception.

Communication between the cytoplasm and the nucleus requires a continuous exchange of molecules across the nuclear envelope (NE). The nuclear pore complex (NPC) is the gateway embedded in the NE through which cargo moves, while transport receptors mediate the passage of macromolecules through the NPC. Although their essential role as the components of the nuclear transport machinery has been extensively studied, how these factors respond to developmental and environmental cues has been underexplored. Here we tag the nucleoporin Nup96 and the transport receptor Imp{beta} with mEGFP and mScarlet-I at their endogenous loci in Drosophila. We demonstrate the functionality of these markers in multiple tissues and offer new options for better visualization of nuclear morphology in densely packed, complex tissues. Then, we characterize the spatiotemporal dynamics of these markers in multiple developmental contexts. We find that Nup96 and Imp{beta} form cytoplasmic puncta, whose size, numbers, and co-localization patterns change dynamically during oogenesis and early embryogenesis. Moreover, we find that the abundance of NPCs per nucleus decreases during early embryogenesis, complementing the emerging model in which NPCs play a regulatory role in development. The tools and observations described here will be useful in understanding the dynamic regulation of nuclear morphology and transport machinery in development.

Border-like cells (BLCs) are sheets of cells that are continuously sloughed off and replenished at the Arabidopsis root cap surface. ROOT CAP POLYGALACTURONASE (RCPG) encodes a putative pectinase involved in BLC shedding. Xylogalacturonan (XGA) is a pectic polysaccharide whose synthesis is associated with cell detachment and secreted separately from other cell wall polysaccharides. BEARSKIN1 (BRN1) and BRN2 are Arabidopsis NAC family transcription factors, and RCPG expression is inhibited in brn1/2. To explore the link between XGA and RCPG, we examined XGA synthesis in Arabidopsis lines with altered RCPG levels. We found that RCPG was contained in XGA-carrying vesicles budding from the trans-Golgi, but XGA synthesis was not affected in the rcpg mutant. XGA was absent in BLCs of brn2, but not of brn1, indicating that BRN2 is necessary for XGA synthesis. Overexpression of functional RCPG-GFP (oeRCPG-GFP) caused upregulation of BRN2, ectopic XGA synthesis, overaccumulation of endogenous RCPG, and accelerated BLC turnover, suggesting a positive regulatory loop between RCPG and BRN2. Inactivation of BRN2 in oeRCPG-GFP suppressed RCPG-GFP expression, excess RCPG, and XGA synthesis. Our data provide evidence that XGA and RCPG are secreted together and that BRN2 controls XGA synthesis, which facilitates RCPG export and BLC separation.

Effectors of the vertebrate Hedgehog (HH) signaling pathway are organized through primary cilia (PC) that grow and retract in lockstep with the cell cycle in response to extracellular signals. Protein kinase A (PKA), a kinase with ubiquitous distribution in most cells, functions as a specific negative regulator of the HH pathway. Its functional specificity in the HH pathway has been suggested to be controlled by cAMP in the PC. However, the regulation of PKA and its functions in PC remain unclear, in part due to the lack of observation of PKA localization in PC during HH resting state as well as conflicting reports of the dynamic changes of cAMP in cilia and HH pathway activity. To address this issue, we have developed a ciliary-localized FRET-based A-kinase activity probe (Nphp3N-AKAR2-CR) as an improved biosensor for monitoring real-time PKA activity in the PC of both cultured cells and living zebrafish embryos. Although the PKA catalytic subunit (PKA-C) was not observed in PC, basal PKA activity in cells could be detected with this probe. In addition, we have found that only ciliary-targeted PKA and not cytosolic PKA, can modulate the HH pathway, even when the integrity of the PC is disrupted. Notably, ciliary PKA activity was barely changed either by inhibition or activation of the HH pathway at the level of Smoothened (SMO), the obligate HH signal transducer. Moreover, we found that even low concentration of the adenylyl cyclase agonist forskolin (FSK) can efficiently inhibit the HH pathway in the presence of the constitutively active variant SMOA1, suggesting that the activation of the HH pathway by SMO may not be due solely to direct regulation of PKA activity in the PC.

Ectopic gene expression is an indispensable tool in biology and medicine, but is often limited by the low efficiency of DNA transfection. We previously reported that depletion of the autophagy receptor p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target for drugs to increase transfection efficiency. To identify such drugs, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the p62-dependent mechanisms, the time required for p62-dependent ubiquitination, which is required for autophagosome formation, was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. Microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405 was markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings demonstrate for the first time that microtubule inhibitors suppress p62 activation as a mechanism for increasing DNA transfection efficiency and provide solutions to increase efficiency.

Transcription of the majority of eukaryotic genes is accompanied by splicing, a process that depends on the assembly of the spliceosome on introns. The timing of spliceosome assembly varies significantly between introns, transcripts, genes and species. While quick co-transcriptional intron removal has been demonstrated for many mammalian genes, most splicing events do not occur immediately after intron synthesis. In this study, we utilized the highly expressed Tg gene, which forms exceptionally long transcription loops (Leidescher et al., 2022), providing a convenient model for studying splicing dynamics using advanced light microscopy. Our single-cell oligopainting-based analysis revealed a delay in splicing several tens of kilobases downstream of a transcribed intron, a finding further supported by standard cell population analyses. We speculate that this phenomenon is due to the abnormally high transcription rate of the Tg gene, which may lead to a localized deficiency in splicing factors and, consequently, delayed spliceosome assembly on thousands of nascent transcripts decorating the gene. Additionally, we found that, in contrast to short introns (<10 kb), long Tg intron (>50 kb) is spliced promptly, providing further support for the idea that intron length may modulate splicing speed.

The centriolar triplet microtubule consists of an A-tubule with 13 protofilaments, and B- and C-tubules, each with 10 protofilaments. Although the formation of the triplets has been shown to require {gamma}-tubulin, its specific role in the formation of each tubule remains elusive. We isolated two novel Chlamydomonas reinhardtii mutants, bld13-1 and bld13-2, each expressing {gamma}-tubulin with a single amino-acid substitution (T292I, E89D, respectively). Similar to known centriole-deficient mutants, both mutants exhibited defects in ciliary assembly, nuclear number, and the number and orientation of cytoplasmic microtubules. Genetic analyses of the mutants, along with expression of the mutant {gamma}-tubulins in the wild-type cells, showed that both mutants exert dominant-negative effects over wild-type {gamma}-tubulin. Interestingly, although the centrioles in these mutants retained the typical nine triplet structure, their triplets frequently lacked several protofilaments in specific regions of the A- and C-tubules. The protofilament loss occurs more frequently at the proximal end of the centriole. These structural defects strongly suggest that {gamma}-tubulin is essential for the stability of the A- and C-tubules of centriolar triplets.

Organelles have unique structures and molecular compositions for their functions and have been classified accordingly. However, many organelles are heterogeneous and in the process of maturation and differentiation. Because traditional methods have a limited number of parameters and spatial resolution, they struggle to capture the heterogeneous landscapes of organelles. Here, we present a method for multi-parametric particle-based analysis of organelles. After disrupting cells, fluorescence microscopy images of organelle particles labeled with six to eight different organelle markers were obtained, and their multi-dimensional data were represented in intuitive two-dimensional UMAP (uniform manifold approximation and projection) spaces. This method enabled visualization of landscapes of seven major organelles as well as the transitional states of endocytic organelles directed to the recycling and degradation pathways. Furthermore, endoplasmic reticulum-mitochondria contact sites were detected in these maps. Our proposed method successfully detects a wide array of organelles simultaneously, enabling the analysis of heterogeneous organelle landscapes.

The primary cilium is a microtubule-based organelle essential for cellular signaling, whose assembly depends on intraflagellar transport (IFT). IFT20, a unique IFT-B component, localizes to both Golgi and cilium/flagellum, suggesting a role in membrane trafficking. Here, we analyzed CrIFT20-mediated ciliary membrane trafficking of channelrhodopsin-1 (ChR1) in Chlamydomonas reinhardtii. Spectroscopic analysis shows recombinant CrIFT20 has a predominantly helical structure and undergoes GTP-dependent shuttle conformational change. Co-immunolocalization of IFT20 in wild-type C. reinhardtii shows puncta distribution throughout cilia and co-localizes directly/indirectly with ChR1 in cilia. Further co-immunoprecipitation supports the cellular biochemical evidence that IFT20 guides the trafficking of ChR1 at the ciliary membrane. Protein interaction network analysis reveals that IFT20 serves as a central adaptor interfacing early ciliary trafficking components with IFT-B complex and BBSome subunits. These findings extend the mechanistic understanding of ciliary membrane protein delivery in C. reinhardtii and reveal a conserved role of IFT20 in photoreceptor trafficking. This study illustrates how coordinated adaptor, motor, and cargo selection maintain the ciliary distribution of ciliary membrane proteins. This study aids in delineating the underlying mechanism of protein trafficking in cilia using C. reinhardtii as a model system, crucial for studying human cilia-associated disorders (ciliopathies).

The reorientation of the Golgi apparatus is crucial for cell migration and is regulated by multi-polarity signals. A number of non-centrosomal microtubules anchor at the surface of the Golgi apparatus and play a vital role in the Golgi reorientation, but how the Golgi are regulated by polarity signals remains unclear. Calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) is a protein that anchors microtubules to the Golgi, a cellular organelle. Our research indicates that CAMSAP2 is dynamically localized at the Golgi during its reorientation processing. Further research shows that CAMSAP2 is potentially regulated by a polarity signaling molecule called MARK2, which interacts with CAMSAP2. We used mass spectrometry to find that MARK2 phosphorylates CAMSAP2 at serine 835, which affects its interaction with the Golgi associated protein USO1 but not with CG-NAP or CLASPs. This interaction is critical for anchoring microtubules to the Golgi during cell migration, altering microtubule polarity distribution, and aiding Golgi reorientation. Our study reveals an important signaling pathway in Golgi reorientation during cell migration, which can provide insights for research in cancer cell migration, immune response, and targeted drug development.

Macroautophagy, a major self-degradation pathway in eukaryotic cells, utilizes autophagosomes to transport self-material to lysosomes for degradation. While microtubular transport is crucial for the proper function of autophagy, the exact roles of factors responsible for positioning autophagosomes remain incompletely understood. In this study, we performed a loss-of-function genetic screen targeting genes potentially involved in microtubular motility. A genetic background that blocks autophagosome-lysosome fusions was used to accurately analyze autophagosome positioning. We discovered that pre-fusion autophagosomes move towards the non-centrosomal microtubule organizing center (ncMTOC) in Drosophila fat cells, which requires a dynein-dynactin complex. This process is regulated by the small GTPases Rab7 and Rab39 together with their adaptors: Epg5 and ema, respectively. The dynein-dependent movement of vesicles toward the nucleus/ncMTOC is essential for efficient autophagosomal fusions with lysosomes and subsequent degradation. Remarkably, altering the balance of kinesin and dynein motors changes the direction of autophagosome movement, indicating a competitive relationship where normally dynein-mediated transport prevails. Since pre-fusion lysosomes were positioned similarly to autophagosomes, it indicates that pre-fusion autophagosomes and lysosomes converge at the ncMTOC, which increases the efficiency of vesicle fusions.