Cell Structure and Function

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.

Jaw1, a tail-anchored protein with 39 carboxyl (C)-terminal amino acids, is oriented to the lumen of the endoplasmic reticulum and outer nuclear membrane. We previously reported that Jaw1, as a member of the KASH protein family, plays a role in maintaining nuclear shape via its C-terminal region. Furthermore, we recently reported that Jaw1 functions as an augmentative effector of Ca2+ release from the endoplasmic reticulum by interacting with the inositol 1,4,5-trisphosphate receptors (IP3Rs). Intriguingly, the C-terminal region is partially cleaved, meaning that Jaw1 exists in the cell in at least two forms: uncleaved and cleaved. However, the mechanism of the cleavage event and its physiological significance remain to be determined. In this study, we demonstrate that the C-terminal region of Jaw1 is cleaved after its insertion by the signal peptidase complex (SPC). Particularly, our results indicate that the SPC with the catalytic subunit SEC11A, but not SEC11C, specifically cleaves Jaw1. Furthermore, using a mutant with a deficit in the cleavage event, we demonstrate that the cleavage event enhances the augmentative effect of Jaw1 on the Ca2+ release ability of IP3Rs. Summary statementThe C-terminal region of Jaw1, a tail-anchored protein, is cleaved by signal peptidase complex and this cleavage event enhances the augmentative effect of Jaw1 on the Ca2+ release activity of inositol 1,4,5-trisphosphate receptors

The measurement of cell activity changes during damage is important to understand the process of cell death and evaluate the effect of medicines. To evaluate cell activity generally, we extended the method of intensity fluctuation in which intensity change in the pixel induced by the movement of organelles was calculated. Cancer, endothelial and iPS cells were damaged by reactive oxygen species (ROS) generated by a fluorescent dye (IR700), hydrogen peroxide, and ultraviolet light. The intensity fluctuation in damaged cells gradually decreased independent of the kind of cell, indicating that the decrease in the fluctuation is a general phenomenon in damaged cells. The rupture of vesicles and mitochondria in the cells were observed upon ROS production. The motility of purified kinesin and dynein which transport vesicles and organelles was inhibited by ROS. These suggest that ROS and cytotoxic molecules spreading from ruptured organelles contribute to the reduction in cell activity which brings about the decrease in the motility and intensity fluctuation of organelles driven by kinesin and dynein.

The uneven distribution of endoplasmic reticulum (ER) underlies the rapid polar growth of pollen tubes. However, the mechanism governing ER distribution remains elusive. In this study, we have identified a pollen tube-specific syntaxin protein, PbrSYP71. Our findings reveal that both overexpression and knocking down of PbrSYP71 inhibited pollen tube growth. Subcellular localization analysis demonstrates that PbrSYP71 anchors to the ER via its transmembrane structure. Overexpression of PbrSYP71 leads to clustered ER distribution in the pollen tube, while knocking down of PbrSYP71 abolishes the uneven ER distribution. Remarkably, transient overexpression of PbrSYP71{Delta}ABD, lacking the actin binding domain (ABD) of PbrSYP71, has no impact on ER distribution or pollen tube growth. Further investigation indicates that ABD is positioned on F-actin in the pollen tube and has a direct interaction with F-actin. PbrSYP71 assists the ER in moving towards the apex of pollen tube, with ABD displaying autonomous mobility. Our study elucidates that PbrSYP71 maintains uneven distribution of the ER by tethering ER to F-actin, facilitating ER movement towards the pollen tube apex for pear pollen tube elongation. These insights shed light on the mechanisms governing ER distribution in polarized cell growth.

Super-resolution microscopic observation of a novel non-toxic sphingomyelin probe revealed the formation of dynamic small domains including sphingomyelin and cholesterol in the cytosolic leaflet of living cell plasma membranes. AbstractSphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.

Mitochondrial biogenesis relies on hundreds of proteins which are derived from genes encoded in the nucleus. According to characteristic properties of N-terminal targeting peptides (TP) and multi-step authentication by the protein translocase called the TOM complex, nascent polypeptides satisfying the requirements are imported into mitochondria. However, it has not been investigated whether the eukaryotic cell with a simple proteome and a single mitochondrion in a cell has a similar or simpler complexity of presequence requirements for mitochondrial protein import as other eukaryotes with multiple mitochondria. Based on the amino acid compositions of putative mitochondrial TP sequences in the unicellular red alga Cyanidioschyzon merolae, we designed the synthetic TP (synTP) and confirmed that synTP-fused mVenus were translocated into the mitochondrion in vivo. Through a series of experimental evaluations using modified synTPs, we showed that functional TP must have some basic residues, at least one, and compose the specific amino acid composition, but the physicochemical properties of net charge, hydrophobicity and hydrophobic moment are not strictly determined in C. merolae. Combined with the simple composition of the TOM complex in C. merolae, our results suggest that a regional positive charge in TP would be recognized and verified solely by TOM22 as a single-step authentication for mitochondrial protein import in C. merolae. The simple authentication mechanism indicates that the C. merolae cell, with its simple cell structure and genome, would not need to increase the cryptographic complexity of the lock-and-key for mitochondrial protein import.

The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death to ensure the survival of organisms. OSW-1, a natural compound with anticancer activity, acts as a potent inhibitor of OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the cholesterol pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed in HeLa cells deficient in factors involved in the PI4P metabolism, such as PITPNB and PI4KB genes. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi apparatus.

- and {beta}-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTT). The function of this region in cilia/flagella is still unclear, except that glutamates in CTT act as the sites for posttranslational modifications that affect ciliary motility. A unicellular alga Chlamydomonas possesses only two -tubulin genes and two {beta}-tubulin genes, each pair encoding an identical protein. This simple gene organization may enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutants expressing tubulins with modified CTTs. We found that the mutant whose four glutamate residues in the -tubulin CTT have been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant lacking the glutamate-rich region of the {beta}-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to the mutants harboring a mutation in a subunit of katanin, whose function has been shown to depend on the {beta}-tubulin CTT. Therefore, our study reveals distinct and important roles of - and {beta}-tubulin CTT in the formation and function of cilia. Summary statementChlamydomonas mutants were produced by CRISPR/Cas9 mediated gene editing to investigate ciliary function of tubulin C-terminal tails (CTTs). We found that - and {beta}-tubulin CTTs are essential for ciliary motility and assembly.

Cyclobutane pyrimidine dimer (CPD) photolyase (PHR), the primary enzyme for repairing the CPD induced by ultraviolet B (UV-B) radiation, is essential for plants living under sunlight. Rice CPD photolyase (OsPHR), is such a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but were yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species and in many other plants, PHR does not localize to chloroplasts Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have acquired this new mechanism for chloroplast translocation.

The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, and filamentous actin (F-actin) forms a variety of types of cellular structures. Many actin probes have been developed to visualize F-actin architectures in eukaryotic cells. However, it is known that double-stained images of F-actin obtained by two different types of actin probes often show partial inconsistencies. While developing actin probes, we observed that the merged images of three distinct actin probes, each labeled with a different fluorescent dye (red, green and blue), enabled various F-actin architectures to be distinguished based on color. This differentiation arises from slight variations in the distribution of each actin probe, and results in a unique color combination of actin probes that reflects the relative intensities of the three fluorescent dyes. In this report, we introduce a new cell staining method, named Actin Painting, which exploits the unique affinity of actin-binding molecules in each actin probe to F-actin architectures. This technique allows the classification of various cell types based on their specific actin cytoskeleton.

Processing bodies (PBs) and stress granules (SGs) are membrane-less cellular compartments consisting of ribonucleoprotein complexes. Whereas PBs are more ubiquitous, SGs are assembled mainly in response to stress. PBs and SGs are known to physically interact and molecules exchange between the two have been documented in mammals. However, the molecular mechanisms underpinning these processes are virtually unknown in plants. We have reported recently that tandem CCCH zinc finger 1 (TZF1) protein can recruit MAPK signaling components to SGs. Here we have found that TZF1-MPK3/6-MKK4/5 form a protein-protein interacting network in SGs. The mRNA decapping factor 1 (DCP1) is a core component of PBs. MAPK signaling mediated phosphorylation triggers a rapid reduction of DCP1 partition into PBs, concomitantly associated with an increase of DCP1 assembly into SGs. Furthermore, we have found that plant SG marker protein UBP1b (oligouridylate binding protein 1b) plays a role in maintaining DCP1 in PBs by suppressing the accumulation of MAPK signaling components. Together, we propose that MAPK signaling and UBP1b mediate the dynamics of PBs and SGs in plant cells.

Fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) to form granules in cells, leading to pathogenic aggregations that cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Proteomics analysis revealed that FUS granules contain valosin-containing protein (VCP), a member of the AAA family ATPase. Confocal microscopy images showed that VCP co-localized in the FUS granules in cells. This study demonstrates that VCP in granules has a two-faced role in FUS granulation: VCP stabilizes de novo FUS granules, while VCP present in the granules for extended periods dissolves them. This VCP function relies on its ATPase activity to consume ATP in granules. VCP stabilizes de novo FUS by reducing intragranular ATP concentrations to a range below the cytosolic concentration. VCP continually consumes ATP during its stay in the granules, which eventually lowers ATP concentrations to a range that destabilizes the granules. VCP, therefore, acts as a timer to limit the residence of FUS granules in cells and thereby prohibits the FUS fibrillization that occurs in persistent granules. VCP ATPase activity plays a role in FUS granule turnover. Summary statementVCP recruited to FUS granules regulates the stability of the granules in a time-dependent manner by consuming intragranular ATP with its ATPase activity.

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.

Type IV P-type ATPases (P4-ATPases) serve as lipid flippases, translocating membrane lipids from the exoplasmic (or luminal) leaflet to the cytoplasmic leaflet of lipid bilayers. In mammals, these P4-ATPases are localized to distinct subcellular compartments. ATP8A1 and ATP9A, both members of the P4-ATPase family, are involved in endosome-mediated membrane trafficking, although the roles of P4-ATPases in the secretory pathway remain to be clarified. ATP9A and ATP9B are located in the trans-Golgi network, with ATP9A also present in endosomal compartments. This study unveiled the overlapping roles of ATP9A and ATP9B in transporting VSVG from the Golgi to the plasma membrane within the secretory pathway. Furthermore, we demonstrated that the flippase activities of ATP9A and ATP9B were crucial for transport process. Notably, we discovered the formation of homomeric and/or heteromeric complexes between ATP9A and ATP9B. The existence of the heteromeric complex notably contributed to the retention of ATP9A in the Golgi. Therefore, ATP9A and ATP9B play a role in the secretory pathway from the Golgi to the plasma membrane, forming either homomeric or heteromeric complexes.

Microbial rhodopsin-based optogenetics has been widely applied to diverse mammalian and plant cell types for controlling membrane potential mediated responses. However, trafficking of optogenetically active protein to the desired subcellular organelle is still a major concern in optogenetic field. This could be resolved by studying the trafficking mechanism of optogenetically active protein in the native system. Current study is focused on the trafficking of two of the microbial rhodopsins named Chalmyopsin5 and Chlamyopsin6 in a green alga, Chlamydomonas reinhardtii. Chlamyopsin5 and Chlamyopsin6 are modular in nature and possess rhodopsin, histidine kinase, response regulator and cyclase domain in tandem. Immunolocalization of Chlamyopsin5 and Chlamyopsin6 in wild strain suggests their different subcellular localization; Chlamyopsin5 in eyespot and Chlamyopsin6 in flagella. Extensive immunocytochemistry of Chlamyopsin5 and Chlamyopsin6 was performed in different intraflagellar transport (IFT) components-defective strains of Chlamydomonas to dissect their trafficking mode to the destined subcellular compartment. Our results indicated the trafficking of Chlamyopsin5 to the eyespot to be independent of IFT machinery while Chlamyopsin6 to the flagella to be IFT dependent. Further, we demonstrate that IFT88 and IFT52 stabilizes Chlamyopsin6 and IFT20 interacts with Chlamyopsin6 in Chlamydomonas. Protein interactome of Chlamyopsin5 and Chlamyopsin6 indicate their role in nitrogen assimilation, gametogenesis and photoprotection in co-ordination with other photoreceptors. Collectively, our study enabled us to understand the targeting of Chlamyopsins to the subcellular compartment (eyespot and flagella). Further research in this direction is required to resolve the current challenge of targeting of optogenetic protein to desired subcellular compartment. HighlightsO_LIChlamyopsin6 localizes in eyespot and flagella of Chlamydomonas reinhardtii and its trafficking is IFT-mediated. C_LIO_LIChlamyopsin5 localizes in the eyespot and its trafficking is independent of IFT machinery. C_LIO_LIDisruption of IFT52 and IFT88 results in reduced Chlamyopsin6 protein content within the cell. C_LIO_LIThe protein interactome suggests the crosstalk among photoreceptors to mediate light-triggered cellular responses. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/693822v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1041d79org.highwire.dtl.DTLVardef@12b06f1org.highwire.dtl.DTLVardef@1d4f970org.highwire.dtl.DTLVardef@1445a3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

To increase our understanding of the inner working of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides and/or signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells. The plasmids are distributed by addgene: https://www.addgene.org/browse/article/28189953/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/160374v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@67f3aaorg.highwire.dtl.DTLVardef@ae2235org.highwire.dtl.DTLVardef@47f729org.highwire.dtl.DTLVardef@b3c9d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Circular dorsal ruffles (CDRs), large-scale rounded membrane ruffles, function as precursors of macropinocytosis. We recently reported that CDRs are exposed in the Hep3B hepatocellular carcinoma cell line, while not in other hepatocellular carcinoma cell lines, indicating that the CDRs in Hep3B are associated with malignant potential. In this study, we investigated the cellular function of CDRs in Hep3B cells by focusing on the molecular mechanisms of the GTPase-activating protein ARAP1. ARAP1 was localized to the CDRs, the sizes of which were reduced by deletion of this protein. High-resolution scanning electron micrographs revealed that CDRs comprise small vertical lamellipodia, the expression pattern of which was disrupted in ARAP1 KO cells. Extracellular solute uptake, rate of cell growth, and malignant potential were attenuated in the KO cells. ARAP1 is also localized in Hep3B cell mitochondria, although not in those of the Huh7 hepatocellular carcinoma cell line. On the basis of these findings, we propose that the aberrant expression of ARAP1 in Hep3B cells modulates CDRs, thereby resulting in an excess uptake of nutrients as an initial event in cancer development. SUMMARY STATEMENTARAP1 regulates circular dorsal ruffles (CDRs) in the Hep3B HCC cell line and deletion of this protein attenuates malignant potential, thereby indicating the involvement of CDRs in cancer development.

Mutations in PKD1 or PKD2 genes lead to autosomal dominant polycystic kidney disease (ADPKD) that is the most frequent family inherited renal disorder. These genes encode polycystin-1/PC-1 and polycycstin-2/PC-2, respectively. Although the genetic basis of ADPKD is well established, the crucial functions of polycystins underlying onset and development of cyst formation remain elusive. Fission yeast Schizosaccharomyces pombe has a single polycystin homolog, Pkd2, which is essential for cell growth. In this study, the truncation analyses of Pkd2 reveal that Pkd2 localizes to not only the plasma membrane but also the endoplasmic reticulum (ER) and regulates cytoplasmic calcium signaling in fission yeast. Internal transmembrane domains within Pkd2 are sufficient for these processes. Surprisingly, more than half of Pkd2 is not required for cell viability. Cytoplasmic calcium levels are mainly regulated through C-terminus of Pkd2. Importantly, human Pkd2 also localizes to the ER and furthermore, fully complements the loss of fission yeast Pkd2. As the functions of polycystin-2 are conserved, fission yeast provides a suitable model to study the mechanism of ADPKD as well as polycystins.