LGN Blocks the Ability of NuMA to Bind and Stabilize Microtubules A Mechanism for Mitotic Spindle Assembly Regulation

AbstractLGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), which is required for polarity establishment and asymmetric cell divisions during embryonic development [1–3]. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis [4–9]. LGN also regulates spindle organization, possibly through inhibition of NuMA function [10], but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, we now show that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. We therefore propose that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization

Multiple Mechanisms Regulate NuMA Dynamics at Spindle Poles

The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. Here, we use both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, we show that an exogenously expressed green-fluorescent-protein/NuMA fusion undergoes continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of approximately 3 minutes. To explore how NuMA dynamics at spindle poles is regulated, we exploited the association of NuMA with microtubule asters formed in mammalian mitotic extracts. Using a monoclonal antibody specific for human NuMA, we followed the fate of human NuMA associated with microtubule asters upon dilution with a hamster mitotic extract. Consistent with in vivo data, this assay shows that NuMA can be displaced from the core of pre-assembled asters into the soluble pool. The half-time of NuMA displacement from asters under these conditions is approximately 5 minutes. Using this assay, we show that protein kinase activity and the NuMA-binding protein LGN regulate the dynamic exchange of NuMA on microtubule asters. Thus, the dynamic properties of NuMA are regulated by multiple mechanisms including protein phosphorylation and binding to the LGN protein, and the rate of exchange between soluble and microtubule-associated pools suggests that NuMA associates with an insoluble matrix at spindle poles

Extremely large magnetoresistance in topologically trivial semimetal α\alpha-WP2_2

Extremely large magnetoresistance (XMR) was recently discovered in many non-magnetic materials, while its underlying mechanism remains poorly understood due to the complex electronic structure of these materials. Here, we report an investigation of the $\alpha$-phase WP$_2$, a topologically trivial semimetal with monoclinic crystal structure (C2/m), which contrasts to the recently discovered robust type-II Weyl semimetal phase in $\beta$-WP$_2$. We found that $\alpha$-WP$_2$ exhibits almost all the characteristics of XMR materials: the near-quadratic field dependence of MR, a field-induced up-turn in resistivity following by a plateau at low temperature, which can be understood by the compensation effect, and high mobility of carriers confirmed by our Hall effect measurements. It was also found that the normalized MRs under different magnetic fields has the same temperature dependence in $\alpha$-WP$_2$, the Kohler scaling law can describe the MR data in a wide temperature range, and there is no obvious change in the anisotropic parameter $\gamma$ value with temperature. The resistance polar diagram has a peanut shape when field is rotated in $\textit{ac}$ plane, which can be understood by the anisotropy of Fermi surface. These results indicate that both field-induced-gap and temperature-induced Lifshitz transition are not the origin of up-turn in resistivity in the $\alpha$-WP$_2$ semimetal. Our findings establish $\alpha$-WP$_2$ as a new reference material for exploring the XMR phenomena.Comment: 18 pages, 12 figure

Par3 Controls Epithelial Spindle Orientation by aPKC-Mediated Phosphorylation of Apical Pins

SummaryBackgroundFormation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, misorientation creates multiple lumens.ResultsWe now show that silencing of the polarity protein Par3 causes spindle misorientation in Madin-Darby canine kidney cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3 or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects.ConclusionsThe Par3 and aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation

Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma

LGN regulates mitotic spindle orientation during epithelial morphogenesis

Disrupting LGN’s function at lateral membrane domains displaces the axis of cell division in cyst-forming MDCK cells

Efficacy and safety of levosimendan in patients with sepsis: a systematic review and network meta-analysis

Objective: We conducted a systematic review to assess the advantages and disadvantages of levosimendan in patients with sepsis compared with placebo, milrinone, and dobutamine and to explore the clinical efficacy of different concentrations of levosimendan.Methods: PubMed, Web of Science, Cochrane Library, Embase, CNKI, Wanfang data, VIP, and CBM databases were searched using such keywords as simendan, levosimendan, and sepsis. The search time was from the establishment of the database to July 2023. Two researchers were responsible for literature screening and data collection respectively. After the risk of bias in the included studies was evaluated, network meta-analysis was performed using R software gemtc and rjags package.Results: Thirty-two randomized controlled trials (RCTs) were included in the network meta-analysis. Meta-analysis results showed that while levosimendan significantly improved CI levels at either 0.1 µg/kg/min (mean difference [MD] [95%CrI] = 0.41 [−0.43, 1.4]) or 0.2 µg/kg/min (MD [95%CrI] =0.54 [0.12, 0.99]). Levosimendan, at either 0.075 µg/kg/min (MD [95% CrI] =0.033 [−0.75, 0.82]) or 0.2 µg/kg/min (MD [95% CrI] = −0.014 [−0.26, 0.23]), had no significant advantage in improving Lac levels. Levosimendan, at either 0.1 µg/kg/min (RR [95% CrI] = 0.99 [0.73, 1.3]) or 0.2 µg/kg/min (RR [95% CrI] = 1.0 [0.88, 1.2]), did not have a significant advantage in reducing mortality.Conclusion: The existing evidence suggests that levosimendan can significantly improve CI and lactate levels in patients with sepsis, and levosimendan at 0.1 µg/kg/min might be the optimal dose. Unfortunately, all interventions in this study failed to reduce the 28-day mortality.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023441220

Two Distinct Integrin-Mediated Mechanisms Contribute to Apical Lumen Formation in Epithelial Cells

Background: Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of b1-integrins in epithelial polarization is well established but the significance of the accompanying a-subunits have not been analyzed in detail. Principal Findings: Here we demonstrate that two distinct integrin-dependent pathways regulate formation of apical lumens to ensure robust apical membrane biogenesis under different microenvironmental conditions; 1) a2b1- and a6b4integrins were required to establish a basal cue that depends on Rac1-activity and guides apico-basal cell polarization. 2) a3b1-integrins were implicated in positioning of mitotic spindles in cysts, a process that is essential for Cdc42-driven epithelial hollowing. Significance: Identification of the separate processes driven by particular integrin receptors clarifies the functional hierarchies between the different integrins co-expressed in epithelial cells and provides valuable insight into the complexity of cell-ECM interactions thereby guiding future studies addressing the molecular basis of epithelial morphogenesis durin