SummaryTo understand further the role of the nucleoid and the min system in selection of the cell division site, we examined FtsZ localization in Escherichia coli cells lacking MinCDE and in parC mutants defective in chromosome segregation. More than one FtsZ ring was sometimes found in the gaps between nucleoids in min mutant filaments. These multiple FtsZ rings were more apparent in longer cells; double or triple rings were often found in the nucleoid-free gaps in ftsI min and ftsA min double mutant filaments. Introducing a parC mutation into the ftsA min double mutant allowed the nucleoid-free gaps to become significantly longer. These gaps often contained dramatic clusters of FtsZ rings. In contrast, filaments of the ftsA parC double mutant, which contained active MinCDE, assembled only one or two rings in most of the large nucleoidfree gaps. These results suggest that all positions along the cell length are competent for FtsZ ring assembly, not just sites at mid-cell or at the poles. Consistent with previous results, unsegregated nucleoids also correlated with a lack of FtsZ localization. A model is proposed in which both the inhibitor y effect of the nucleoid and the regulation by MinCDE ensure that cells divide precisely at the midpoint.

FtsZ, a tubulin‐like GTPase that forms a dynamic ring marking the division plane of prokaryotic cells, is essential for cytokinesis. It is not known what triggers FtsZ ring assembly. In this work, we use a FtsZ–green fluorescent protein (Gfp) chimera to assay FtsZ assembly over time by using fluorescence microscopy. We show that FtsZ polymers can assemble dynamically in solution in a GTP‐dependent manner. Initially, FtsZ nucleation centers grow into aster‐like structures that dramatically resemble microtubule organizing centers. As assembly proceeds further, protofilament bundles emanating from different asters interconnect, mimicking the closure of the FtsZ ring in vivo. Surprisingly, millimolar levels of Ca2+ promote FtsZ dynamic assembly. FtsZ can undergo repeated GTP‐dependent assembly and disassembly in solution by sequential addition and removal of Ca2+. In addition, GTP binding and hydrolysis by FtsZ are regulated by Ca2+ concentration. Although the concentration of Ca2+ required for FtsZ assembly in vitro is high, its clear and specific effect on FtsZ dynamics suggests the possibility that Ca2+ may have a role in regulating FtsZ ring assembly in the cell.

SARS-CoV-2 has been reported to show a capacity for invading the brains of humans and model animals. However, it remains unclear whether and how SARS-CoV-2 crosses the blood–brain barrier (BBB). Herein, SARS-CoV-2 RNA was occasionally detected in the vascular wall and perivascular space, as well as in brain microvascular endothelial cells (BMECs) in the infected K18-hACE2 transgenic mice. Moreover, the permeability of the infected vessel was increased. Furthermore, disintegrity of BBB was discovered in the infected hamsters by administration of Evans blue. Interestingly, the expression of claudin5, ZO-1, occludin and the ultrastructure of tight junctions (TJs) showed unchanged, whereas, the basement membrane was disrupted in the infected animals. Using an in vitro BBB model that comprises primary BMECs with astrocytes, SARS-CoV-2 was found to infect and cross through the BMECs. Consistent with in vivo experiments, the expression of MMP9 was increased and collagen IV was decreased while the markers for TJs were not altered in the SARS-CoV-2-infected BMECs. Besides, inflammatory responses including vasculitis, glial activation, and upregulated inflammatory factors occurred after SARS-CoV-2 infection. Overall, our results provide evidence supporting that SARS-CoV-2 can cross the BBB in a transcellular pathway accompanied with basement membrane disrupted without obvious alteration of TJs.

Two members of the angiopoietin-like family of proteins, ANGPTL3 and ANGPTL4, have been shown to play important roles in modulating lipoprotein metabolism in the body. Both proteins were found to suppress lipoprotein lipase (LPL) activity in vitro as well as in vivo. However, their mechanisms of inhibition remained poorly understood. Using enzyme kinetic analysis with purified recombinant proteins, we have found key mechanistic differences between ANGPTL3 and ANGPTL4. ANGPTL3 reduced LPL catalytic activity but did not significantly alter its self-inactivation rate. In contrast, ANGPTL4 suppressed LPL by accelerating the irreversible inactivation of LPL. Furthermore, heparin was able to overcome the inhibitory effect of ANGPTL3 on LPL but not that of ANGPTL4. Site-directed mutagenesis demonstrated the critical function of Glu 40 in ANGPTL4. In contrast, when cysteine residues involved in disulfide bond formation were mutated to serines, ANGPTL4 retained its activity. Taken together, our data provide a more detailed view of the structure and mechanisms of these proteins. The finding that ANGPTL3 and ANGPTL4 inhibit LPL activity through distinct mechanisms indicates that the two proteins play unique roles in modulation of lipid metabolism in vivo. Lipoprotein lipase (LPL)2 is an essential enzyme that catalyzes the hydrolysis of triglycerides to generate free fatty acids and monoacylglycerol (for review, see Refs. 1 and 2). It is synthesized and secreted by adipocytes, macrophages, and muscle cells and then bound to the vascular endothelium by heparin sulfate proteoglycans and GPIHBP1 (glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1), a recently discovered protein (3, 4). LPL anchored as such releases free fatty acids and monoacylglycerol from triglycerides carried by chylomicron and very low density lipoprotein particles (5-9) and thus plays a major role in lipid metabolism. LPL-deficient subjects have severe hypertriglyceridemia and increased risk of arteriosclerosis (10). In contrast, subjects with slightly increased LPL activity were found to have lower triglyceride levels and decreased risk of cardiovascular diseases (11).It is well known that LPL is rapidly inactivated in vivo, but the underlying mechanism is unknown (12, 13). Recently, two secreted proteins were found to inhibit LPL activity both in vitro and in vivo. These two proteins, known as ANGPTL3 and ANGPTL4, are members of the angiopoietin-like protein family (14 -17). They share 31% overall sequence homology, with an N-terminal domain containing a coiled-coil region and a C-terminal fibrinogen-like domain that is cleaved off in vivo (16,18). Both proteins are found to inhibit LPL activity in vitro (16,18). Overexpression of ANGPTL3 and ANGTPL4 in mice led to extremely high blood levels of triglycerides and cholesterol (15, 19 -21). Knock-out of either gene in mice results in much lower blood levels of these lipids (14,(17)(18)(19)(22)(23)(24). Furthermore, post-heparin plasma LPL activity is significantly elevated in...