Cell structure and function最新文献

Cyclin-dependent kinases (CDKs) orchestrate cell cycle progression through precise temporal control of substrate phosphorylation. While traditional biochemical approaches and phosphoproteomics have provided valuable insights into CDK-mediated regulation, these methods require cell population analyses and cannot capture real-time dynamics in individual cells. The recent development of fluorescent biosensors has revolutionized our ability to monitor CDK activity in living cells with unprecedented temporal and spatial resolution. Here, we comprehensively review genetically encoded fluorescent biosensors for measuring CDK activity. The two major modes of action in CDK activity biosensors-FRET-based and translocation-based biosensors-enable researchers to select appropriate tools for their specific experimental objectives. These biosensors have revealed precise spatiotemporal CDK activity dynamics across diverse model systems, including yeast, cultured mammalian cells, worms, flies, frog egg extract, fish, and mice. Such technological advances are transforming our understanding of quantitative principles underlying cell cycle control and opening new avenues for investigating cell cycle regulation in various biological contexts.Key words: CDK, FRET, cell cycle, live imaging, biosensor.

Newly synthesized proteins destined for the secretory pathway are folded and assembled in the endoplasmic reticulum (ER) and then transported to the Golgi apparatus via COPII vesicles, which are normally 60-90 nm. COPII vesicles must accordingly be enlarged to accommodate proteins larger than 90 nm, such as long-chain collagen. Key molecules involved in this enlargement are Tango1 and Tango1-like (Tali), which are transmembrane proteins in the ER encoded by the MIA3 and MIA2 genes, respectively. Interestingly, two splicing variants are expressed from each of these two genes: Tango1L and Tango1S from the MIA3 gene, and Tali and cTAGE5 from the MIA2 gene. Here, we constructed Tango1L-knockout (KO), Tango1S-KO, Tali-KO, and cTAGE5-KO separately in medaka fish, a vertebrate model organism, and characterized them. Results showed that only Tango1L-KO conferred a lethal phenotype to medaka fish. Only Tango1L-KO medaka fish exhibited a shorter tail than wild-type (WT) fish and showed the defects in the export of type II collagen from the ER, contrary to the previous reports analyzing Tango1-KO or Tali-KO mice and the results of knockdown experiments in human cultured cells. Medaka fish may employ a simpler system than mammals for the export of large molecules from the ER.Key words: intracellular transport, COPII vesicles, enlargement, endoplasmic reticulum, Golgi apparatus.

The process of mammalian myogenesis is fundamental to understanding muscle development and holds broad relevance across multiple fields, from developmental biology to regenerative medicine. This review highlights two key aspects: myoblast proliferation and the role of cilia in this process. Myoblasts, as muscle precursor cells, must undergo tightly regulated cycles of proliferation and differentiation to ensure proper muscle growth and function. Recent research has uncovered an essential role for primary cilia, hair-like sensory organelles on the cell surface, in modulating signaling pathways crucial to myogenesis. Cilium-mediated signaling appears to regulate various stages of myogenesis, including the control of myoblast differentiation. Furthermore, primary cilia undergo multiple cycles of formation and disassembly during myogenesis, presumably enabling detailed, context-dependent regulation of their functions. In particular, the regulation of myoblast proliferation through cell cycle control by primary cilia is an important topic that requires further investigation. By examining the interactions between primary cilia and myoblasts, this review aims to provide new insights into the molecular and cellular mechanisms driving muscle development, with potential applications for understanding muscle-related diseases and advancing therapeutic strategies. Additionally, advancements in imaging and image analysis technologies have become indispensable for studying these processes at the cellular level. This review also addresses these technological advancements and current challenges.Key words: myogenesis, myoblast, proliferation, cilia, imaging.

The motility of biological molecular motors has typically been analyzed by in vitro reconstitution systems using motors isolated and purified from organs or expressed in cultured cells. The behavior of biomolecular motors within cells has frequently been reported to be inconsistent with that observed in reconstituted systems in vitro. Although this discrepancy has been attributed to differences in ionic strength and intracellular crowding, understanding how such parameters affect the motility of motors remains challenging. In this report, we investigated the impact of intracellular crowding in vitro on the mechanical properties of kinesin under a high ionic strength that is comparable to the cytoplasm. Initially, we characterized viscosity in a cell by using a kinesin motor lacking the cargo-binding domain. We then used polyethylene glycol to create a viscous environment in vitro comparable to the intracellular environment. Our results showed that kinesin frequently dissociated from microtubules under high ionic strength conditions. However, under conditions of both high ionic strength and crowding with polymers, the processive movement of kinesin persisted and increased in frequency. This setting reproduces the significant variations in the mechanical properties of motors measured in the intracellular environment and suggests a mechanism whereby kinesin maintains motility under the high ionic strengths found in cells.Key words: kinesin motility, molecular crowding, ionic strength, intracellular transport, processivity of molecular motors.

Membrane stiffness is essential for cell migration, tumorigenesis, and development; however, the physical properties of intracellular membrane are poorly characterized. In this study, we internalized 20 nm magnetic nanoparticles (MNPs) into MCF7 human breast cancer cells and applied a magnetic field. We investigated whether magnetic field could induce membrane damage of the early endosomes by analyzing the colocalization of MNPs with galectin 3 (Gal3), a cytosolic protein recruited to the lumen of damaged organelles. We first tried to apply magnetic field by electromagnet, and found a direct-current (DC) magnetic field for five minutes increased the colocalization of the MNPs with Gal3, suggesting that the magnetic field damaged the endosomal membrane. We used a neodymium magnet to apply longer and stronger static magnetic fields. The static magnetic field more than 50 mT for five minutes started to damage endosomes, while 100 mT was the most effective. Longer exposure or higher magnetic field strengths did not induce further membrane damage. We confirmed that a Gal3 positive compartment was also positive for the early endosome marker, EEA1, suggesting that the external magnetic field induced membrane damage in the early endosomes. Our results indicate that a static magnetic field can control the membrane damage in early endosomes using internalized MNPs.Key words: magnetic nanoparticles, endosomes, membrane damage, organelle.

Extracellular signal-regulated kinase (ERK) regulates multiple cellular functions through distinct activation patterns. Genetically encoded fluorescent probes are instrumental in dissecting the ERK activity dynamics in living cells. Here we modified a previously reported Förster resonance energy transfer (FRET) probe for ERK, EKAREN5 by replacing its mTurquoise2 and YPet sequences with mTurquoise-GL and a synonymous codon variant of YPet, respectively. The modified biosensor, EKAREN5-gl, showed an increased sensitivity to EGF-induced ERK activation responding to a very low dose (20 pg/ml) of EGF stimulation. We quantitatively characterized two FRET-based ERK probes, EKAREN5 and EKAREN5-gl, and a subcellular kinase translocation-based probe, ERK-KTR. We found the three biosensors differently respond to EGF stimulations with different intensity, duration, and latency. Furthermore, we investigated how the minimal EGF-induced ERK activation affects the downstream transcription in HeLa cells by comprehensive transcriptional analysis. We found the minimal ERK activation leads to a distinct transcriptional pattern from those induced by higher ERK activations. Our study highlights the significance of sensitive fluorescent probes to understand cellular signal dynamics and the role of minimal ERK activation in regulating transcription.Key words: fluorescent probe, ERK, FRET, KTR.

Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.

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.Key words: fluorescence imaging, organelle, cytoskeleton, small GTPase, Dictyostelium.

Anhydrobiosis, a phenomenon in which organisms survive extreme dehydration by entering a reversible ametabolic state, is a remarkable example of survival strategies. This study focuses on anhydrobiosis in tardigrades, which are known for their resilience to severe environmental conditions. Tardigrades utilize several protective mechanisms against desiccation, notably the constitutive expression of cytoplasmic abundant heat soluble (CAHS) proteins in Ramazzottius varieornatus. These proteins share similarities in their amphiphatic alpha helices with late embryogenesis abundant (LEA) proteins, but differ significantly in their amino acid sequences. In this study, we further explored the functionality of CAHS proteins by analyzing their role in aggregation and tolerance to hyperosmotic stress in mammalian cells. Using live cell imaging, we examined the subcellular localization of several CAHS and LEA proteins in response to hyperosmotic stress. The expression of CAHS1, CAHS3, and CAHS8 tended to enhance the resilience to the hyperosmotic conditions. These findings not only deepen our understanding of the molecular mechanisms of anhydrobiosis but also highlight the potential of CAHS proteins as cryoprotectants.Key words: anhydrobiosis, Tardigrades, live imaging, disordered proteins, desiccation tolerance.

The sarcomere is the contractile unit of striated muscle and is composed of actin and myosin filaments. There is increasing evidence to support that actin assembly mediated by Fhod3, a member of the formin family of proteins, is critical for sarcomere formation and maintenance in cardiac muscle. Fhod3, which is abundantly expressed in the heart, localizes to the center of sarcomeres and contributes to the regulation of the cardiac function, as evidenced by the fact that mutations in Fhod3 cause cardiomyopathy. However, the role of Fhod3 in skeletal muscle, another type of striated muscle, is unclear. We herein show that Fhod3 is expressed in the tongue at both mRNA and protein levels, although in smaller amounts than in the heart. To determine the physiological role of Fhod3 expressed in the tongue, we generated embryos lacking Fhod3 in the tongue. The tongue tissue of the Fhod3-depleted embryos did not show any significant structural defects, suggesting that Fhod3 is dispensable for normal development of the mouse tongue. Unexpectedly, the immunostaining analysis revealed the absence of specific sarcomeric signals for Fhod3 in the wild-type tongue when compared to the Fhod3-depleted tongue as a negative control, despite the use of antibodies that had previously been validated by immunostaining of heart tissues. Taken together, although Fhod3 protein is expressed at a significant level in the tongue, Fhod3 in the tongue does not appear to exhibit the same sarcomeric pattern as observed in the heart, suggesting a different role for Fhod3 in the tongue muscles.Key words: actin, formin, sarcomere, striated muscle.