Simulation examining the factors influencing capillary wick transport in a refrigerant direct cooling system for power battery packs.

The effectiveness of power battery refrigerant direct cooling systems of electric vehicles incorporating capillary wicks is directly determined by these wicks' transport performance. The Fries-Dreyer equation describes wicking behavior, but there is a significant gap between its predictions and the experimental results as reported in the literature. This work examines the factors influencing transport performance in an unconsolidated capillary wick with spherical particles. A mathematical and physical model is developed, the latter using the COMSOL software platform. Both the developed mathematical form and the numerically simulated results of this model are closer to the experimental results than those obtained using the Fries-Dreyer equation. The simulation results enable optimizing the equilibrium height and capillary time numbers providing a fitted Fries-Dreyer equation that is then used to analyze the influence of saturation, inclination angle, wick particle diameter, and tortuosity on the liquid rise mass and velocity and the equilibrium height, and the effects are in close but not perfect accord with experimental data. To narrow the gap, the Fries-Dreyer equation is further optimized using the numerically simulated results, substantially improving the accord with the experimental results.

Regulation of mTORC2 Signaling.

Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase and a master regulator of cell growth and metabolism, forms two structurally and functionally distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. While mTORC1 signaling is well characterized, mTORC2 is relatively poorly understood. mTORC2 appears to exist in functionally distinct pools, but few mTORC2 effectors/substrates have been identified. Here, we review recent advances in our understanding of mTORC2 signaling, with particular emphasis on factors that control mTORC2 activity.

The ionization process of chemical warfare agent simulants in low temperature plasma ionization.

The application of low-temperature plasma ionization technology in the chemical warfare agent detection was mostly focused on the research of rapid detection methods. Limited studies are available on the ionization process of chemical warfare agents in low temperature plasma. Through the intensity of protonated molecules of dimethyl methylphosphonate (DMMP) in different solvents including methanol, deuterated methanol (methanol-D4), pure water, and deuterium oxide (water-D2), it was concluded that the water molecule in the air provides the hydrogen ion (H+) needed for ionization. The product ion spectra and the collision-induced dissociation processes of protonated molecules of nerve agent simulants, including DMMP, diethyl methanephosphonate (DEMP), trimethyl phosphate (TMP), triethyl phosphate (TEP), tripropyl phosphate (TPP), and tributyl phosphate (TBP) were analyzed. Results revealed that H+ mostly combined with phosphorus oxygen double bond (P = O) in the low-temperature plasma ionization. By analyzing the peak intensity distribution of product ions of protonated molecules, the presence of proton and charge migration in the low temperature plasma ionization and collision-induced dissociation were researched. This study could provide technical guidance for the rapid and accurate detection of chemical warfare agents through low temperature plasma ionization-mass spectrometry.

A distal centriolar protein network controls organelle maturation and asymmetry.

A long-standing mystery in the centrosome field pertains to the origin of asymmetry within the organelle. The removal of daughter centriole-specific/enriched proteins (DCPs) and acquisition of distal appendages on the future mother centriole are two important steps in the generation of asymmetry. We find that DCPs are recruited sequentially, and their removal is abolished in cells lacking Talpid3 or C2CD3. We show that removal of certain DCPs constitutes another level of control for distal appendage (DA) assembly. Remarkably, we also find that Talpid3 forms a distal centriolar multi-functional hub that coordinates the removal of specific DCPs, DA assembly, and recruitment of ciliary vesicles through distinct regions mutated in ciliopathies. Finally, we show that Talpid3, C2CD3, and OFD1 differentially regulate the assembly of sub-distal appendages, the CEP350/FOP/CEP19 module, centriolar satellites, and actin networks. Our work extends the spatial and functional understanding of proteins that control organelle maturation and asymmetry, ciliogenesis, and human disease.

RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor ß signaling.

The activity of the E3 ligase, SMURF2, is antagonized by an intramolecular, autoinhibitory interaction between its C2 and Hect domains. Relief of SMURF2 autoinhibition is induced by TGFß and is mediated by the inhibitory SMAD, SMAD7. In a proteomic screen for endomembrane interactants of the RING-domain E3 ligase, RNF11, we identified SMURF2, among a cohort of Hect E3 ligases previously implicated in TGFß signaling. Reconstitution of the SMURF2·RNF11 complex in vitro unexpectedly revealed robust SMURF2 E3 ligase activity, with biochemical properties previously restricted to the SMURF2·SMAD7 complex. Using in vitro binding assays, we find that RNF11 can directly compete with SMAD7 for SMURF2 and that binding is mutually exclusive and dependent on a proline-rich domain. Moreover, we found that co-expression of RNF11 and SMURF2 dramatically reduced SMURF2 ubiquitylation in the cell. This effect is strictly dependent on complex formation and sorting determinants that regulate the association of RNF11 with membranes. RNF11 is overexpressed in certain tumors, and, importantly, we found that depletion of this protein down-regulated gene expression of several TGFß-responsive genes, dampened cell proliferation, and dramatically reduced cell migration in response to TGFß. Our data suggest for the first time that the choice of binding partners for SMURF2 can sustain or repress TGFß signaling, and RNF11 may promote TGFß-induced cell migration.

Role for the IFT-A Complex in Selective Transport to the Primary Cilium.

Intraflagellar transport sub-complex A (IFT-A) is known to regulate retrograde IFT in the cilium. To rigorously assess its other possible roles, we knocked out an IFT-A subunit, IFT121/WDR35, in mammalian cells and screened the localization of more than 50 proteins. We found that Wdr35 regulates cilium assembly by selectively regulating transport of distinct cargoes. Beyond its role in retrograde transport, we show that Wdr35 functions in fusion of Rab8 vesicles at the nascent cilium, protein exit from the cilium, and centriolar satellite organization. Furthermore, we show that Wdr35 is essential for entry of many membrane proteins into the cilium through robust interactions with cargoes and other IFT-A subunits, but the actin network functions to dampen this transport. Wdr35 is mutated in several ciliopathies, and we find that certain disease mutations impair interactions with cargo and other IFT-A subunits. Together, our data link defects in IFT-A mediated cargo transport with disease.

Primary cilia control hedgehog signaling during muscle differentiation and are deregulated in rhabdomyosarcoma.

The primary cilium acts as a cellular antenna, transducing diverse signaling pathways, and recent evidence suggests that primary cilia are important in development and cancer. However, a role for cilia in normal muscle development and rhabdomyosarcoma (RMS) has not been explored. Here we implicate primary cilia in proliferation, hedgehog (Hh) signaling, and differentiation of skeletal muscle cells. Cilia and Hh signaling are highly dynamic during the differentiation of myoblasts. We show that cilia are assembled during the initial stages of myogenic differentiation but disappear as cells progress through myogenesis, concomitant with the destruction of proteins critical for cilia assembly and shortly after the Hh effector, Gli3, leaves the cilium. Importantly, we show that ablation of primary cilia strongly suppresses Hh signaling and myogenic differentiation while enhancing proliferation. Interestingly, our data further indicate that both cilia assembly and Hh signaling are deregulated in RMS, and cilia respond to Hh ligand in certain subsets of RMS cells but not others. Together, these findings provide evidence for an essential role for both primary cilia assembly and disassembly in the control of Hh signaling and early differentiation in muscle cells. We suggest that the temporally orchestrated destruction of centrosomal and ciliary proteins is a necessary antecedent for removal of the primary cilium and cessation of Hh signaling during myogenic differentiation. Additionally, our results further stratify RMS populations and highlight cilia assembly and disassembly as potential RMS drug targets.

Self-assembly and sorting of acentrosomal microtubules by TACC3 facilitate kinetochore capture during the mitotic spindle assembly.

Kinetochore capture by dynamic kinetochore microtubule fibers (K fibers) is essential for proper chromosome alignment and accurate distribution of the replicated genome during cell division. Although this capture process has been extensively studied, the mechanisms underlying the initiation of this process and the proper formation of the K fibers remain largely unknown. Here we show that transforming acidic coiled-coil-containing protein 3 (TACC3) is essential for kinetochore capture and proper K-fiber formation in HeLa cells. To observe the assembly of acentrosomal microtubules more clearly, the cells were released from higher concentrations of nocodazole into zero or lower concentrations. We find that small acentrosomal TACC3-microtubule aster formation near the kinetochores and binding of the asters with the kinetochores are the initial steps of the kinetochore capture by the acentrosomal microtubules, and that the sorting of kinetochore-captured acentrosomal microtubules with centrosomal microtubules leads to the capture of kinetochore by centrosomal microtubules from both spindle poles. We demonstrate that the sorting of the TACC3-associated microtubules with the centrosomal microtubules is a crucial process for spindle assembly and chromosome movement. These findings, which are also supported in the unperturbed mitosis without nocodazole, reveal a critical TACC3-dependent acentrosomal microtubule nucleation and sorting process to regulate kinetochore-microtubule connections and provide deep insight into the mechanisms of mitotic spindle assembly and chromosome alignment.

USP33 regulates centrosome biogenesis via deubiquitination of the centriolar protein CP110.

Centrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. Centrosome duplication is controlled in part by CP110, a centriolar protein that positively regulates centriole duplication while restricting centriole elongation and ciliogenesis. Maintenance of normal CP110 levels is essential, as excessive CP110 drives centrosome over-duplication and suppresses ciliogenesis, whereas its depletion inhibits centriole amplification and leads to highly elongated centrioles and aberrant assembly of cilia in growing cells. CP110 levels are tightly controlled, partly through ubiquitination by the ubiquitin ligase complex SCF(cyclin F) during G2 and M phases of the cell cycle. Here, using human cells, we report a new mechanism for the regulation of centrosome duplication that requires USP33, a deubiquitinating enzyme that is able to regulate CP110 levels. USP33 interacts with CP110 and localizes to centrioles primarily in S and G2/M phases, the periods during which centrioles duplicate and elongate. USP33 potently and specifically deubiquitinates CP110, but not other cyclin-F substrates. USP33 activity antagonizes SCF(cyclin F)-mediated ubiquitination and promotes the generation of supernumerary centriolar foci, whereas ablation of USP33 destabilizes CP110 and thereby inhibits centrosome amplification and mitotic defects. To our knowledge, we have identified the first centriolar deubiquitinating enzyme whose expression regulates centrosome homeostasis by countering cyclin-F-mediated destruction of a key substrate. Our results point towards potential therapeutic strategies for inhibiting tumorigenesis associated with centrosome amplification.

Novel functions of endocytic player clathrin in mitosis.

Clathrin has been widely recognized as a pivotal player in endocytosis, in which several adaptors and accessory proteins are involved. Recent studies suggested that clathrin is also essential for cell division. Here this review mainly focuses on the clathrin-dependent mechanisms involved in spindle assembly and chromosome alignment. In mitosis, clathrin forms a complex with phosphorylated TACC3 to ensure spindle stability and proper chromosome alignment. The clathrin-regulated mechanism in mitosis requires the crosstalk among clathrin, spindle assembly factors (SAFs), Ran-GTP and mitotic kinases. Meanwhile, a coordinated mechanism is required for role transitions of clathrin during endocytosis and mitosis. Taken together, the findings of the multiple functions of clathrin besides endocytosis have expanded our understanding of the basic cellular activities.

Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment.

Transforming acidic coiled-coil-containing protein 3 (TACC3) has been implicated in mitotic spindle assembly, although the mechanisms involved are largely unknown. Here we identify that clathrin heavy chain (CHC) binds specifically to phosphorylated TACC3 and recruits it to spindle poles for proper spindle assembly and chromosome alignment. Phosphorylation of Xenopus TACC3 at serine 620 (S620) and S626, but not S33, is required for its binding with CHC. Knockdown of CHC by RNA interference (RNAi) abolishes the targeting of TACC3 to spindle poles and results in abnormal spindle assembly and chromosome misalignment, similar to the defects caused by TACC3 knockdown. Furthermore, the binding of CHC with phosphorylated TACC3 is inhibited by importin ß and this inhibition is reversed by the presence of the GTP-binding nuclear protein Ran in the GTP-bound state. Together, these results indicate that the recruitment of phosphorylated TACC3 to spindle poles by CHC ensures proper spindle assembly and chromosome alignment, and is regulated by Ran.