Kangmin He

Long-distance intercellular connectivity between cardiomyocytes and cardiofibroblasts mediated by membrane nanotubes

AIMS Intercellular interactions between cardiomyocytes (CMs) and cardiofibroblasts (FBs) are important in the physiological and pathophysiological heart. Understanding such interactions is important for developing effective heart disease therapies. However, until recently, little has been known about these interactions. We aimed to investigate structural and functional connections between CMs and FBs that are distinct from gap junctions. METHODS AND RESULTS By membrane dye staining, we observed long, thin membrane nanotubular structures containing actin and microtubules that connected neonatal rat ventricular CMs and FBs. By single-particle tracking, we observed vehicles moving between CMs and FBs within the membrane nanotubes. By dual colour staining, confocal imaging and flow cytometry, we observed mitochondria exchange between CMs and FBs in a coculture system. By combined atomic force microscopy (AFM) and confocal microscopy, we observed calcium signal propagation from AFM-stimulated CM (or FB) to unstimulated FB (or CM) via membrane nanotubes. By membrane and cytoskeleton staining, we observed similar nanotubular structures in adult mouse heart tissue, which suggests their physiological relevance. CONCLUSIONS As a novel type of CM to FB communication, membrane nanotubes observed in vitro and in vivo provide structural and functional connectivity between CMs and FBs over long distances.

Intercellular transportation of quantum dots mediated by membrane nanotubes.

In this work, we reported that the quantum dot (QD) nanoparticles could be actively transported in the membrane nanotubes between cardiac myocytes. Single particle imaging and tracking of QDs revealed that most QDs moved in a bidirectional mode along the membrane nanotubes with a mean velocity of 1.23 mum/s. The results suggested that QDs moving in the nanotubes were coordinately motivated by molecular motors. It provides new information for the study of intercellular transportation of nanoparticles.

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes.

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling.

Single-molecule imaging revealed enhanced dimerization of transforming growth factor β type II receptors in hypertrophic cardiomyocytes.

Transforming growth factor β (TGF-β) signaling plays an important role in the pathogenesis of cardiac hypertrophy. However, the molecular mechanism of TGF-β signaling during the process of cardiac remodeling remains poorly understood. In the present study, by employing single-molecule fluorescence imaging approach, we demonstrated that in neonatal rat cardiomyocytes, TGF-β type II receptors (TβRII) existed as monomers at the low expression level, and dimerized upon TGF-β1 stimulation. Importantly, for the first time, we found the increased dimerization of TβRII in hypertrophic cardiomyocytes comparing to the normal cardiomyocytes. The enhanced TβRII dimerization was correlated with the enhanced Smad3 phosphorylation levels. These results provide new information on the mechanism of TGF-β signaling in cardiac remodeling.

Mitochondria are transported along microtubules in membrane nanotubes to rescue distressed cardiomyocytes from apoptosis

Membrane nanotubes (MNTs) act as “highways” between cells to facilitate the transfer of multiple signals and play an important role in many diseases. Our previous work reported on the transfer of mitochondria via MNTs between cardiomyocytes (CMs) and cardiac myofibroblasts (MFs); however, the elucidation of the underlying mechanism and pathophysiological significance of this transfer requires additional study. In this study, we determined that the mean movement velocity of mitochondria in MNTs between CMs and MFs was approximately 17.5 ± 2.1 nm/s. Meanwhile, treatment with microtubule polymerisation inhibitors nocodazole or colcemid in cell culture decreased mitochondrial velocity, and knockdown of the microtubule motor protein kinesin family member 5B (KIF5B) led to a similar effect, indicating that mitochondrial movement was dependent on microtubules and the motor protein KIF5B. Furthermore, we showed that hypoxia/reoxygenation-induced CM apoptosis was attenuated by coculture with intact or hypoxia/reoxygenation-treated MFs, which transferred mitochondria to CMs. This rescue was prevented either by separating the cells using Transwell culture or by impairing mitochondrial transfer with nocodazole or colcemid treatment. In conclusion, as a novel means of intercellular communication, MNTs rescue distressed CMs from apoptosis by transporting mitochondria along microtubules via KIF5B.

Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

Single-molecule imaging reveals the stoichiometry change of epidermal growth factor receptor during transactivation by β 2 -adrenergic receptor

Stimulation of G protein-coupled receptors (GPCRs) can lead to the transactivation of the epidermal growth factor receptors (EGFR). The cross-communication between the two signaling pathways regulates several important physiological or pathological processes. However, the molecule mechanism underlying EGFR transactivation remains poorly understood. Here, we aim to study the GPCR-mediated EGFR transactivation process using the single-molecule fluorescence imaging and tracking approach. We found that although EGFR existed as monomers at the plasma membrane of resting cells, they became dimers and thus diffused slower following the activation of β2-adrenergic receptor (β2-AR) by isoproterenol (ISO). We further proved that β2-AR-mediated changes of EGFR in stoichiometry and dynamics were mediated by Src kinase. Thus, the observations obtained via the single-molecule imaging and tracking methods shed new insights into the molecular mechanism of EGFR transactivation at single molecule level.

Mammalian actin-binding protein 1/HIP-55 is essential for the scission of clathrin-coated pits by regulating dynamin-actin interaction

Actin and dynamin work cooperatively to drive the invagination and scission of clathrin‐coated pits (CCPs). However, little is known about the mechanism that orchestrates the spatiotemporal recruitment of dynamin and actin. Here, we have identified the mammalian actin‐binding protein 1 (mAbp1; also called HIP‐55 or SH3P7), which could bind to clathrin, actin, as well as dynamin, as an adaptor that links the dynamic recruitment of dynamin and actin for the scission of CCPs. Live‐cell imaging reveals that mAbp1 is specifically recruited at a late stage of the long‐lived CCPs. mAbp1 knockdown impaired CCP scission by reducing dynamin recruitment at the plasma membrane. However, actin disruption remarkably eliminates mAbp1 recruitment and thus dynamin recruitment. These data suggest that by binding to both clathrin and F‐actin, mAbp1 is specifically recruited at a late stage of CCP formation, which subsequently recruits dynamin to CCPs. He, K., Xing, R., Yan, X., Tian, A., Zhang, M., Yuan, J., Lv, Z., Fang, X., Li, Z., and Zhang, Y.—Mammalian actin‐binding protein 1/HIP‐55 is essential for the scission of clathrin‐coated pits by regulating dynamin‐actin interaction. FASEB J. 29, 2495‐2503 (2015). www.fasebj.org

Quantitative Characterization of the Membrane Dynamics of Newly Delivered TGF-β Receptors by Single-Molecule Imaging

The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-β type II receptor (TβRII), a typical serine/threoninekinase receptor, was studied with this method. We first eliminated the fluorescence signals from the pre-existing EGFP-labeled TβRII molecules on the plasma membrane, and then we recorded the individual newly appeared TβRII-GFP by total-internal-reflection fluorescence imaging. The fluorescence-intensity distributions, photobleaching steps, and diffusion rates of the single TβRII-GFP molecules were analyzed. We reported, for the first time, that TβRII was transported to the plasma membrane mainly in the monomeric form in both resting and TGF-β1stimulated cells. This strongly supported our former discovery that TβRII could exist as a monomer on the cell membrane. We also found that ligand stimulation resulted in enhanced delivery rates and prolonged membrane-association times for the TβRII molecules. On the basis of these observations, we proposed a mechanism of TGF-β1-induced TβRII dimerization for receptor activation. Our method provides a useful tool for the real-time quantification of the spatial arrangement, mobility, and oligomerization of cell-surface proteins in living cells, thus providing a better understanding of cell signaling.