Probing CENP-E function in chromosome dynamics using small molecule inhibitor syntelin

Dear Editor, Chromosome movements during mitosis are orches- trated primarily by the interaction of spindle microtu- bules with the kinetochore [1], the site for attachment of spindle microtubules to the centromere. The kinetochore has an active function in chromosomal segregation through microtubule-based motors located at or near it [1-2]. CENP-E is a microtubule-based kinesin motor [3],

Mesoporous MnSiO3@Fe3O4@C Nanoparticle as p H-responsive T1-T2* Dual-modal Magnetic Resonance Imaging Contrast Agent for Tumor Diagnosis

Mesoporous structured MnSiO3@Fe3O4@C nanoparticles(NPs)were prepared via a facile and efficient strategy,with negligible cytotoxicity and minor side efforts.The as-prepared MnSiO3@Fe3O4@C NPs hold great potential in serving as pH-responsive T1-T2^*dual-modal magnetic resonance(MR)imaging contrast agents.The released Mn^2+shortened T1 relaxation time,meanwhile the superparamagnetic Fe3O4 enhanced T2 contrast imaging.The release rate of Mn ions reaches 31.66%under the condition of pH=5.0,which is similar to tumor microenvironment and organelles.Cytotoxicity assays show that MnSiO3@Fe3O4@C NPs have minor toxicity,even at high concentrations.After intravenous injection of MnSiO3@Fe3O4@C NPs,a rapid contrast enhancement in tumors was achieved with a significant enhancement of 132%after 24 h of the administration.Moreover,a significant decreasement of 53.8%was witnessed in T2 MR imaging signal.It demonstrated that MnSiO3@Fe3O4@C NPs can act as both positive and negative MR imaging contrast agents.Besides,owing to the pH-responsive degradation of mesoporous MnSiO3,MnSiO3@Fe3O4@C NPs can also be used as potential drug systems for cancer theranostics.

Organization of microtubule plus-end dynamics by phase separation in mitosis

In eukaryotes,microtubule polymers are essential for cellular plasticity and fate decisions.End-binding(EB)proteins serve as scaffolds for orchestrating microtubule polymer dynamics and are essential for cellular dynamics and chromosome segregation in mitosis.Here,we show that EB1 forms molecular condensates with TIP150 and MCAK through liquid–liquid phase separation to compartmentalize the kinetochore–microtubule plus-end machinery,ensuring accurate kinetochore–microtubule interactions during chromosome segregation in mitosis.Perturbation of EB1–TIP150 polymer formation by a competing peptide prevents phase separation of the EB1-mediated complex and chromosome alignment at the metaphase equator in both cultured cells and Drosophila embryos.Lys220 of EB1 is dynamically acetylated by p300/CBP-associated factor in early mitosis,and persistent acetylation at Lys220 attenuates phase separation of the EB1-mediated complex,dissolves droplets in vitro,and harnesses accurate chromosome segregation.Our data suggest a novel framework for understanding the organization and regulation of eukaryotic spindle for accurate chromosome segregation in mitosis.

Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death

Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer （NK） cells enter target tumor ceils, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internalization and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we car- ried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Impor- tantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.

Gd Doped Hollow Nanoscale Coordination Polymers as Multimodal Imaging Agents and a Potential Drug Delivery Carriers

Gd doped hollow nanoscale coordination polymers with multimodal imaging capabilities were synthesized by solvothermal method and further coated by silica layer.The in vitro tests demonstrated uncoated and silica-coated nanoprobes exhibit longitudinal relaxivities(r1)of 7.38 and 13.57(mmol/L)-1·s-1,and transverse relaxivities(r2)of 180.6 and 304.8(mmol/L)-1·s-1,showing fairly good dual T1&T2 contrast effects,and it also emits excellent multicolor uorescence under laser beams of various wavelengths.With the com-bination of magnetic resonance imaging(MRI)(both T1 and T2)and uorescence optical imaging(FOI),the nanoprobes could correlate preoperative diagnosis with intraoperative pathology.Furthermore,it also exhibits high drug loading capacity of 1166 mg/g and en-capsulation efficiency of 83.29%,which makes it a potential platform as drug carriers.The MTT assay demonstrates the moderate toxicity of the NPs,and after the silica coating process,not only the MRI contrast effects but also the biocompatibility have been enhanced.The versatility of the highly integrated systems can make up for the limitations of each imaging modality and exhibit great potentials for cancer theranostics.

PML–LRIF1 interactions form a novel link between promyelocytic leukemia bodies and centromeres

Dear Editor,Promyelocytic leukemia(PML)is the scaffold protein that organizes PML bod-ies,which are nuclear membraneless organelles involved in various biologi-cal processes,including tumor suppres-sion and antiviral responses(Ugge et al.,2022).Early electron microscopic analy-ses revealed contacts between the sur-face of PML bodies and chromatin struc-ture(Corpet et al.,2020).In fact,sev-eral chromatin and cell cycle regulators,such as TIP60,P300,and heterochro-matin protein 1(HP1),are localized in PML bodies in interphase cells(Corpet et al.,2020).

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis

Stable transmission of genetic information during cell division requires faithful chromosome segregation.Mounting evidence has demonstrated that polo-like kinase 1(PLK1)dynamics at kinetochores control correct kinetochore–microtubule attachments and subsequent silencing of the spindle assembly checkpoint.However,the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive.Here,we identified a regulatory mechanism by which PLK1-elicited zeste white 10(ZW10)phosphorylation regulates spindle checkpoint silencing in mitosis.ZW10 is a cognate substrate of PLK1,and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10–Zwint1 interactions.Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes,while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase,in which sister chromatids entangled as cells entered anaphase.These findings reveal the previously uncharacterized PLK1–ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.

Aurora B promotes the CENP-T–CENP-W interaction to guide accurate chromosome segregation in mitosis

Accurate chromosome segregation in mitosis depends on kinetochores that connect centromeric chromatin to spindle microtubules.Centromeres are captured by individual microtubules via a kinetochore constitutive centromere-associated network(CCAN)during chromosome segregation.CCAN contains 16 subunits,including CENP-W and CENP-T.However,the molecular recognition and mitotic regulation of the CCAN assembly remain elusive.Here,we revealed that CENP-W binds to the histone fold domain and an uncharacterized N-terminal region of CENP-T.Aurora B phosphorylates CENP-W at threonine 60,which enhances the interaction between CENP-W and CENP-T to ensure robust metaphase chromosome alignment and accurate chromosome segregation in mitosis.These findings delineate a conserved signaling cascade that integrates protein phosphorylation with CCAN integrity for the maintenance of genomic stability.

CSPP1 stabilizes microtubules by capping both plus and minus ends

Although the dynamic instability of microtubules(MTs)is fundamental to many cellular functions,quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics.However,how these free MT tips are stabilized remains poorly understood.Here,we report that centrosome and spindle pole protein 1(CSPP1)caps and stabilizes both plus and minus ends of static MTs.Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends,whose dynamic instability was concomitantly suppressed.Consistently,MT ends in CSPP1-overexpressing cells were hyper-stabilized,while those in CSPP1-depleted cells were much more dynamic.This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting polymerization.Importantly,CSPP1-bound MTs were resistant to mitotic centromere-associated kinesin-mediated depolymerization.These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

Mitosis-specific acetylation tunes Ran effector binding for chromosome segregation

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. The Ran GTPase plays a key role in mitotic spindle assembly. However, how the generation of a chemical gradient of Ran-GTP at the spindle is coupled to mitotic post-translational modifications has never been characterized. Here, we solved the complex structure of Ran with the nucleotide release factor Mogl and delineated a novel mitosis-specific acetylation-regulated Ran-Mogl interaction dur- ing chromosome segregation. Our structure-guided functional analyses revealed that Mogl compotes with RCCl for Ran binding in a GTP/GDP-dependent manner. Biochemical characterization demonstrated that Mogl-bound Ran prevents RCCl binding and subse- quent GTP loading. Surprisingly, Ran is a bono fide substrate of TIP60, and the acetylation of Lys134 by TIP60 liberates Mogl from Ran binding during mitosis. Importantly, this acetylation-elicited switch of Ran binding to RCC1 promotes high level of Ran-GTP, which is essential for chromosome alignment. These results establish a previously uncharacterized regulatory mechanism in which TIP60 pro- vides a homeostatic control of Ran-GTP level by tuning Ran effector binding for chromosome segregation in mitosis.

Phosphorylation of CENP-R by Aurora B regulates kinetochore-microtubule attachment for accurate chromosome segregation

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes.Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis.Although previously proposed to be an adaptor of retinoic acid receptor,here,we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and ensure accurate chromosome segregation in mitosis.We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore,suggesting that phosphorylation may regulate its localization.Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore-microtubule attachment at metaphase.Mechanistically,CENP-R phosphorylation disrupts its binding with CENP-U.Thus,we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore-microtubule attachment in mitosis.As CENP-R is absent from yeast,we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.

Dynamic phosphorylation of CENP-N by CDK1 guides accurate chromosome segregation in mitosis

In mitosis,accurate chromosome segregation depends on the kinetochore,a supermolecular machinery that couples dynamic spin-dle microtubules to centromeric chromatin.However,the structure–activity relationship of the constitutive centromere-associated network(CCAN)during mitosis remains uncharacterized.Building on our recent cryo-electron microscopic analyses of human CCAN structure,we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation.Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1,which modulates the CENP-L–CENP-N interaction for accurate chromosome segregation and CCAN organization.Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint.These analyses provide mechanistic insight into a previously undefined link between the centromere–kinetochore network and accurate chromosome segregation.

Sgo1 interacts with CENP-A to guide accurate chromosome segregation in mitosis

Shugoshin-1(Sgo1)is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis.It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120.However,it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis.Here,we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis.Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A.Interestingly,the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere,resulting in aberrant chromosome segregation and premature chromatid separation.Taken together,these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1–CENP-A interaction guides accurate cell division.

LRIF1 interacts with HPla to coordinate accurate chromosome segregation during mitosis

Heterochromatin protein 1α (HP1α)regulates chromatin specification and plasticity during cell fate decision.Different structural determinants account for HP1α Localization and function during cell division cycle.Our earlier study showed that centromeric Localization of HP1α depends on the epigenetic mark H3K9me3 in interphase,while its centromeric location in mitosis relies on uncharacterized PXVXL-containing factors.Here,we identified a PXVXL-containing protein,Ligand-dependent nuclear receptorinteracting factor 1 (LRIF1),which recruits HPla to the centromere of mitotic chromosomes and its interaction with HP1α is essential for accurate chromosome segregation during mitosis.LRIF1 interacts directly with HPla chromoshadow domain via an evolutionariLy conserved PXVXL motif within its C-terminus.Importantly,the LRIF1-HPla interaction is critical for Aurora B activity in the inner centromere.Mutation of PXVXL motif of LRIF1 Leads to defects in HPla centromere targeting and aberrant chromosome segregation.These findings reveal a previously unrecognized direct Link between LRIF1 and HP1α in centromere plasticity control and illustrate the critical role of LRIF1-HP1α interaction in orchestrating accurate cell division.

Acetytation of ACAP4 regutates CCL18-elicited breast cancer cell migration and invasion

Tumor metastasis represents the main causes of cancer-related death.Our recent study showed that chemokine CCL18 secreted from tumor-associated macrophages regulates breast tumor metastasis,but the underlying mechanisms remain less clear.Here, we show that ARF6 GTPase-activating protein ACAP4 regulates CCL18-elicited breast cancer cell migration via the acetyltransferase PCAF-mediated acetylation.CCL18 stimulation elicited breast cancer cell migration and invasion via PCAF-dependent acetylation.ACAP4 physically interacts with PCAF and is a cognate substrate of PCAF during CCL18 stimulation.The acetylation site of ACAP4 by PCAF was mapped to Lys311 by mass spectrometric analyses.Importantly,dynamic acetylation of ACAP4 is essential for CCL18-induced breast cancer cell migration and invasion,as overexpression of the persistent acetylation-mimicking or nonacetylatable ACAP4 mutant blocked CCL18-elicited cell migration and invasion.Mechanistically,the acetylation of ACAP4 at Lys311 reduced the lipid-binding activity of ACAP4 to ensure a robust and dynamic cycling of ARF6-ACAP4 complex with plasma membrane in response to CCL18 stimulation.Thus,these results present a previously undefined mechanism by which CCL18-elicited acetylation of the PH domain controls dynamic interaction between ACAP4 and plasma membrane during breast cancer cell migration and invasion.

SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules.Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complexand spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internaldynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP cometsin vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora Bactivity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.

Acetylation of Nup62 by TIP60 ensures accurate chromosome segregation in mitosis

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation.In eukaryotic cells,nuclear envelope breakdown(NEBD)is required for proper chromosome segregation.Although a list of mitotic kinases has been implicated in NEBD,how they coordinate their activity to dissolve the nuclear envelope and protein machinery such as nuclear pore complexes was unclear.Here,we identified a regulatory mechanism in which Nup62 is acetylated by TIP60 in human cell division.Nup62 is a novel substrate of TIP60,and the acetylation of Lys432 by TIP60 dissolves nucleoporin Nup62-Nup58-Nup54 complex during entry into mitosis.Importantly,this acetylation-elicited remodeling of nucleoporin complex promotes the distribution of Nup62 to the mitotic spindle,which is indispensable for orchestrating correct spindle orientation.Moreover,suppression of Nup62 perturbs accurate chromosome segregation during mitosis.These results establish a previously uncharacterized regulatory mechanism in which TIP60-elicited nucleoporin dynamics promotes chromosome segregation in mitosis.

Alp7-Mtol and Alpl4 synergize to promote interphase microtubule regrowth from the nuclear envelope

Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers(MTOCs),including the nuclear envelope(NE)and pre-existing microtubules.The evolutionarily conserved proteins Mtol/CDK5RAP2 and Alpl4/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation.However,it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs,particularly the NE,and how these proteins coordinate to organize microtubule assembly.Here,we demonstrate that in the fission yeast Schizosaccharomyces pombe,efficient interphase microtubule growth from the NE requires Alp7/TACC,Alpl4/TOG/XMAP215,and Mtol/CDK5RAP2.The absence of Alp7,A lp l4 t or Mtol compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization.We further demonstrate that Alp7 and Mtol interdependently localize to the NE in cells without microtubules and that A lp l4 localizes to the NE in an Alp7 and Mtol-dependent manner.Tethering Mtol to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE.Hence,our study delineates that Alp7,A lpl4,and Mtol work in concert to regulate interphase microtubule regrowth on the NE.

Cdk1:conductor of CENP-A symphony orchestra

The inheritance of human genome through mitosis is pre- cisely regulated by a series of tightly orchestrated events such as chromosome condensation, bi-orientated spindle formation, chromosome congression, segregation and cytokinesis. Chromosome movements during mitosis are governed by dynamic interactions of spindle microtubules with a specialized chromosome domain called centromere. Centromere provides the platform for the assembly of a protein machine named kinetochore that orchestrates accurate interaction between chromosomes and spindle microtubules during cell division. In eukaryotic organisms, the centromere is epigenetically specified by nucleosomes containing a unique histone H3 variant CENP-A, with the exception of budding yeast [1]. CENP-A orthologues present in all known eukaryotes. It is the most important epigenetic marker of centromere [2] and is required for the correct localization of most centromere- and kinetochore- related proteins [2]. CENP-A is the epigenetic marker to maintain and propagate the centromere identity, which assembles into a nucleosome together with histories H4, H2A and H2B without any known specific selection of DNA sequence [1, 2]. Centromeric chromatin is inter- spersed with CENP-A-nucleosomes and histone H3-con- taining nucleosomes, and it forms a repressive heterochromatin structure through the concerted action of various epigenetic mechanisms including histone methyl- ation [2].The plasticity of centromeric chromatin is crucial for the assembly of a large protein machine kinetochore that links chromosome to the spindle microtubule for mitotic segregation.

Measurement of dynamic membrane mechanosensation using optical tweezers

Many cellular processes are orchestrated by dynamic changes in the plasma membrane to form membrane projections and endocytic compartments in response to extracellular cue changes.Our previous studies show that post-translational modifications of ACAP4 regulate membrane dynamics and curvature in response to epidermal growth factor and chemokine(C−C motif)ligand 18 stimulation(Zhao et al.,2013;Song et al.,2018).