Stay in touch with the endoplasmic reticulum.

The endoplasmic reticulum (ER), which is composed of a continuous network of tubules and sheets, forms the most widely distributed membrane system in eukaryotic cells. As a result, it engages a variety of organelles by establishing membrane contact sites (MCSs). These contacts regulate organelle positioning and remodeling, including fusion and fission, facilitate precise lipid exchange, and couple vital signaling events. Here, we systematically review recent advances and converging themes on ER-involved organellar contact. The molecular basis, cellular influence, and potential physiological functions for ER/nuclear envelope contacts with mitochondria, Golgi, endosomes, lysosomes, lipid droplets, autophagosomes, and plasma membrane are summarized.

NuMA regulates mitotic spindle assembly, structural dynamics and function via phase separation.

A functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.

K6-linked SUMOylation of BAF regulates nuclear integrity and DNA replication in mammalian cells.

Barrier-to-autointegration factor (BAF) is a highly conserved protein in metazoans that has multiple functions during the cell cycle. We found that BAF is SUMOylated at K6, and that this modification is essential for its nuclear localization and function, including nuclear integrity maintenance and DNA replication. K6-linked SUMOylation of BAF promotes binding and interaction with lamin A/C to regulate nuclear integrity. K6-linked SUMOylation of BAF also supports BAF binding to DNA and proliferating cell nuclear antigen and regulates DNA replication. SENP1 and SENP2 catalyze the de-SUMOylation of BAF at K6. Disrupting the SUMOylation and de-SUMOylation cycle of BAF at K6 not only disturbs nuclear integrity, but also induces DNA replication failure. Taken together, our findings demonstrate that SUMOylation at K6 is an important regulatory mechanism that governs the nuclear functions of BAF in mammalian cells.

Postmitotic annulate lamellae assembly contributes to nuclear envelope reconstitution in daughter cells.

In higher eukaryotic cells, the nuclear envelope (NE) is composed of double nuclear membranes studded with nuclear pore complexes (NPCs) and undergoes dynamic disassembly and reassembly during the cell cycle. However, how the NE and NPC reassemble remains largely unclear. Here, using HeLa, HEK293, and Drosophila cells, along with immunofluorescence microscopy and transmission EM methods, we found that postmitotic annulate lamellae (AL) assembly contributes to NE and NPC assembly. We observed that the AL are parallel membrane-pair stacks and possess regularly spaced AL pore complexes (ALPCs) that are morphologically similar to the NPCs. We found that the AL assemble in the cytoplasm during mitotic exit simultaneously with NE re-formation in daughter cells. Then, the assembled AL either bound the decondensing chromatin to directly transform into the NE or bound and fused with the outer nuclear membrane to join the assembling NE. The AL did not colocalize with sheet and tubular endoplasmic reticulum (ER) marker proteins on the ER or the lamin B receptor-localized membrane in the cytoplasm, suggesting that postmitotic AL assembly occurs independently of the chromatin and ER. Collectively, our results indicate that postmitotic AL assembly is a common cellular event and an intermediate step in NE and NPC assembly and in NE expansion in higher eukaryotic cells.

The Plk1 kinase negatively regulates the Hedgehog signaling pathway by phosphorylating Gli1.

Hedgehog (Hh) signaling is a highly conserved cell signaling pathway important for cell life, development and tumorigenesis. Increasing evidence suggests that the Hh signaling pathway functions in certain phases of the cell cycle. However, the coordination between Hh signaling and cell cycle control remains poorly understood. Here, we show that polo-like kinase-1 (Plk1), a critical protein kinase regulating many processes during the cell cycle, also regulates Hh signaling by phosphorylating and inhibiting Gli1, a downstream transcription factor of the Hh signaling pathway. Gli1 expression increases along with Hh signaling activation, leading to upregulation of Hh target genes, including cyclin E, during the G1 and S phases. Gli1 is phosphorylated at S481 by Plk1, and this phosphorylation facilitates the nuclear export and binding of Gli1 with its negative regulator Sufu, leading to a reduction in Hh signaling activity. Inhibition of Plk1 kinase activity led to Gli1 maintaining is role in promoting downstream gene expression. Collectively, our data reveal a novel mechanism regarding the crosstalk between Hh signaling and cell cycle control.

Heterogeneous dimer peptide-conjugated polylysine dendrimer-Fe3O4 composite as a novel nanoscale molecular probe for early diagnosis and therapy in hepatocellular carcinoma.

A novel nanoscale molecular probe is formulated in order to reduce toxicity and side effects of antitumor drug doxorubicin (DOX) in normal tissues and to enhance the detection sensitivity during early imaging diagnosis. The mechanism involves a specific targeting of Arg-Gly-Asp peptide (RGD)-GX1 heterogeneous dimer peptide-conjugated dendrigraft poly-l-lysine (DGL)-magnetic nanoparticle (MNP) composite by αvß3-integrin/vasculature endothelium receptor-mediated synergetic effect. The physicochemical properties of the nanoprobe were characterized by using transmission electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction, dynamic light scattering (DLS), and vibrating sample magnetometer. The average diameter of the resulting MNP-DGL-RGD-GX1-DOX nanoparticles (NPs) was ~150-160 nm by DLS under simulate physiological medium. In the present experimental system, the loading amount of DOX on NPs accounted for 414.4 mg/g for MNP-DGL-RGD-GX1-DOX. The results of cytotoxicity, flow cytometry, and cellular uptake consistently indicated that the MNP-DGL-RGD-GX1-DOX NPs were inclined to target HepG2 cells in selected three kinds of cells. In vitro exploration of molecular mechanism revealed that cell apoptosis was associated with the overexpression of Fas protein and the significant activation of caspase-3. In vivo magnetic resonance imaging and biodistribution study showed that the MNP-DGL-RGD-GX1-DOX formulation had high affinity to the tumor tissue, leading to more aggregation of NPs in the tumor. In vivo antitumor efficacy research verified that MNP-DGL-RGD-GX1-DOX NPs possessed significant antitumor activity and the tumor inhibitory rate reached 78.5%. These results suggested that NPs could be promising in application to early diagnosis and therapy in hepatocellular carcinoma as a specific nanoprobe.