Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex.

During cell division, NuMA orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules, and other cellular factors. Here we used in vitro reconstitution, cryo-electron microscopy, and live cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein/dynactin/NuMA complex (DDN) and showed that the NuMA N-terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells.

HURP regulates Kif18A recruitment and activity to synergistically control microtubule dynamics.

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP.

Molecular interplay between HURP and Kif18A in mitotic spindle regulation.

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging , we demonstrate that Kif18A motility is regulated by HURP.

Molecular interplay between HURP and Kif18A in mitotic spindle regulation.

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging , we demonstrate that Kif18A motility is regulated by HURP.

Dyneins.

Dyneins are a family of motor proteins that carry out motility and force generation functions towards the minus end of microtubule filaments. Cytoplasmic dynein (dynein-1) is responsible for transporting intracellular cargos in the retrograde direction in the cytoplasm, anchoring several organelles to the microtubule network, driving nuclear migration in developing neurons, and orienting the mitotic spindle in dividing cells. All other dyneins are localized to cilia.

Nde1 promotes Lis1-mediated activation of dynein.

Cytoplasmic dynein drives the motility and force generation functions towards the microtubule minus end. The assembly of dynein with dynactin and a cargo adaptor in an active transport complex is facilitated by Lis1 and Nde1/Ndel1. Recent studies proposed that Lis1 relieves dynein from its autoinhibited conformation, but the physiological function of Nde1/Ndel1 remains elusive.

Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules.

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro.

Development of a Ratiometric Tension Sensor Exclusively Responding to Integrin Tension Magnitude in Live Cells.

Integrin tensions are critical for cell mechanotransduction. By converting force to fluorescence, molecular tension sensors image integrin tensions in live cells with a high resolution. However, the fluorescence signal intensity results collectively from integrin tension magnitude, tension dwell time, integrin density, sensor accessibility, and so forth, making it highly challenging to specifically monitor the molecular force level of integrin tensions.

Nde1 Promotes Lis1-Mediated Activation of Dynein.

Cytoplasmic dynein is the primary motor that drives the motility and force generation functions towards the microtubule minus end. The activation of dynein motility requires its assembly with dynactin and a cargo adaptor. This process is facilitated by two dynein-associated factors, Lis1 and Nde1/Ndel1.

Ubiquitous membrane-bound DNase activity in podosomes and invadopodia.

Podosomes and invadopodia, collectively termed invadosomes, are adhesive and degradative membrane structures formed in many types of cells and are well known for recruiting various proteases. However, another major class of degradative enzymes, deoxyribonuclease (DNase), remains unconfirmed and not studied in invadosomes. Here, using surface-immobilized nuclease sensor (SNS), we demonstrated that invadosomes recruit DNase to their core regions, which degrade extracellular double-stranded DNA.

Cell Migration Driven by Self-Generated Integrin Ligand Gradient on Ligand-Labile Surfaces.

Integrin-ligand interaction mediates the adhesion and migration of many metazoan cells. Here, we report a unique mode of cell migration elicited by the lability of integrin ligands. We found that stationary cells spontaneously turn migratory on substrates where integrin ligands are subject to depletion by cellular force.

Cellular Force Nanoscopy with 50 nm Resolution Based on Integrin Molecular Tension Imaging and Localization.

Integrin-transmitted cellular forces have rich spatial dynamics and are vital to many cellular functions. To advance the sensitivity and spatial resolution of cellular force imaging, we developed a force-activatable emitter reporting single-molecular tension events and the associated cellular force nanoscopy (CFN). Immobilized on a surface, the emitters are initially dark (>99.

Peptide nucleic acid based tension sensor for cellular force imaging with strong DNase resistance.

DNA is a versatile biomaterial with well-defined mechanical and biochemical properties. It has been broadly adopted to synthesize tension sensors that calibrate and visualize cellular forces at the cell-matrix interface. Here we showed that DNA-based tension sensors are vulnerable to deoxyribonucleases (DNases) which cells may express on cell membrane or secret to the culture environment.

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor.

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level.

Optical sensor revealed abnormal nuclease spatial activity on cancer cell membrane.

Nucleases are important enzymes that cleave nucleic acids and play critical roles in DNA repair, immune defense and potentially in cancer invasion. However, their spatial dynamics at subcellular level is much less studied. Here, we developed a surface-tethered nuclease sensor (SNS) which directly converts membrane-bound nuclease (MN) activity to fluorescent signal, therefore, mapping MN activity on cell adhesion sites with high resolution and sensitivity.

Keratocytes Generate High Integrin Tension at the Trailing Edge to Mediate Rear De-adhesion during Rapid Cell Migration.

Rapid cell migration requires efficient rear de-adhesion. It remains undetermined whether cells mechanically detach or biochemically disassemble integrin-mediated rear adhesion sites in highly motile cells such as keratocytes. Using molecular tension sensor, we calibrated and mapped integrin tension in migrating keratocytes.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging.

Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.

Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets.

Glycine protects against high sucrose and high fat-induced non-alcoholic steatohepatitis in rats.

We set out to explore the hypothesis that glycine attenuates non-alcoholic steatohepatitis (NASH) in rats and the possible mechanism by which is it does. Male Sprague-Dawley (SD) rats were fed a diet containing high fat and high sucrose (HSHF) for 24 weeks to induce NASH. Blood and liver tissues were sampled at selected time points throughout the study.

A model of metabolic syndrome and related diseases with intestinal endotoxemia in rats fed a high fat and high sucrose diet.

Aim: We sought develop and characterize a diet-induced model of metabolic syndrome and its related diseases.

Methods: The experimental animals (Spague-Dawley rats) were randomly divided into two groups, and each group was fed a different feed for 48 weeks as follows: 1) standard control diet (SC), and 2) a high sucrose and high fat diet (HSHF). The blood, small intestine, liver, pancreas, and adipose tissues were sampled for analysis and characterization.

[Changes in mast cells and hepatic expression of c-kit and stem cell factor in the rat model of chronic hepatitis].

Objective: To study the potential role of mast cells and the related molecular mechanism in chronic hepatitis (CH) using a rat model system.

Methods: Thirty Wistar rats (15 males, 15 females; weight range: 230-290 g) were randomly divided into the normal contrast (NC) group and experimental CH group. The CH group received subcutaneous injection of CCl4 and a diet high in cholesterol and alcohol content and low in protein and choline content.

Effects of glycine on phagocytosis and secretion by Kupffer cells in vitro.

Aim: To investigate the effects and mechanisms of action of glycine on phagocytosis and tumor necrosis factor (TNF)-α secretion by Kupffer cells in vitro.

Methods: Kupffer cells were isolated from normal rats by collagenase digestion and Percoll density gradient differential centrifugation. After culture for 24 h, Kupffer cells were incubated in fresh Dulbecco's Modification of Eagle's Medium containing glycine (G1: 1 mmol/L, G2: 10 mmol/L, G3: 100 mmol/L and G4: 300 mmol/L) for 3 h, then used to measure phagocytosis by a bead test, TNF-α secretion after lipopolysaccharide stimulation by radioactive immunoassay, and microfilament and microtubule expression by staining with phalloidin-fluorescein isothiocyanate (FITC) or a monoclonal anti-α tubulin-FITC antibody, respectively, and evaluated under a ultraviolet fluorescence microscope.

Dynamic changes and mechanism of intestinal endotoxemia in partially hepatectomized rats.

Aim: To explore the mechanism of intestinal endo-toxemia (IETM) formation and its changes in partially hepatectomized (PH) rats.

Methods: One-hundred and two adult male Wistar rats were randomly divided into three groups: normal control (NC) group, partially hepatectomized (PH) group and a sham-operated (SO) group. To study the dynamic changes, rats were sacrificed before and at different time points after partial hepatectomy or the sham-operation ( 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 120 h and 168 h).

Experimental study on the role of endotoxin in the development of hepatopulmonary syndrome.

Aim: To evaluate the role of intestinal endotoxemia in the genesis of hepatopulmonary syndrome.

Methods: A rat model of cirrhosis was prepared with the method of compound factors. At the end of the eighth week, rats with cirrhosis were treated with 300 microg LPS/100 g body weight, and 1 g/rat of glycine about four h prior to LPS.

[Effect of glycine on the expression of CD(14) gene and protein of hepatic tissue in the course of developing cirrhosis of rats].

Objective: To observe the effect of glycine on the expression of CD(14) mRNA and protein of hepatic tissue in the course of developing cirrhosis of rats.

Methods: The cirrhotic model of Wistar rats was established by complex pathogens, who were respectively fed with control diets and control diets adding glycine (1g/d, giving by intragastric infusion) or 5% glycine containing diets at the same time. The rats were sacrificed at 2, 4, and 8 weeks, respectively.