Faithful genome propagation requires coordination between nuclear envelope (NE) breakdown, spindle formation, and chromosomal events. The conserved linker of nucleoskeleton and cytoskeleton (LINC) ...complex connects fission yeast centromeres and the centrosome, across the NE, during interphase. During meiosis, LINC connects the centrosome with telomeres rather than centromeres. We previously showed that loss of telomere-LINC contacts compromises meiotic spindle formation. Here, we define the precise events regulated by telomere-LINC contacts and address the analogous possibility that centromeres regulate mitotic spindle formation. We develop conditionally inactivated LINC complexes in which the conserved SUN-domain protein Sad1 remains stable but severs interphase centromere-LINC contacts. Strikingly, the loss of such contacts abolishes spindle formation. We pinpoint the defect to a failure in the partial NE breakdown required for centrosome insertion into the NE, a step analogous to mammalian NE breakdown. Thus, interphase chromosome-LINC contacts constitute a cell-cycle control device linking nucleoplasmic and cytoplasmic events.
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•Mitotic and meiotic spindle formation require prior chromosome-LINC contact•Sad1.2 maintains LINC stability but destroys interphase centromere-LINC contact•Partial nuclear envelope breakdown is lost in sad1.2 cells•Chromosome-LINC interactions control spindle via nuclear envelope regulation
The role of interphase centromere-LINC contacts has been a longstanding intractable mystery. Fernández-Álvarez et al. generate a mutation that severs these centromere-LINC contacts, leading to failed nuclear envelope breakdown and, in turn, failed spindle assembly. Thus, centromere-LINC contacts couple chromosomal events with cytoplasmic events that propel the cell cycle.
Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. ...Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte–nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart.
Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly ...present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.
Germline and somatic genomes are in general the same in a multicellular organism. However, programmed DNA elimination leads to a reduced somatic genome compared to germline cells. Previous work on ...the parasitic nematode Ascaris demonstrated that programmed DNA elimination encompasses high-fidelity chromosomal breaks and loss of specific genome sequences including a major tandem repeat of 120 bp and ~1,000 germline-expressed genes. However, the precise chromosomal locations of these repeats, breaks regions, and eliminated genes remained unknown. We used PacBio long-read sequencing and chromosome conformation capture (Hi-C) to obtain fully assembled chromosomes of Ascaris germline and somatic genomes, enabling a complete chromosomal view of DNA elimination. We found that all 24 germline chromosomes undergo comprehensive chromosome end remodeling with DNA breaks in their subtelomeric regions and loss of distal sequences including the telomeres at both chromosome ends. All new Ascaris somatic chromosome ends are recapped by de novo telomere healing. We provide an ultrastructural analysis of Ascaris DNA elimination and show that eliminated DNA is incorporated into double membrane-bound structures, similar to micronuclei, during telophase of a DNA elimination mitosis. These micronuclei undergo dynamic changes including loss of active histone marks and localize to the cytoplasm following daughter nuclei formation and cytokinesis where they form autophagosomes. Comparative analysis of nematode chromosomes suggests that chromosome fusions occurred, forming Ascaris sex chromosomes that become independent chromosomes following DNA elimination breaks in somatic cells. These studies provide the first chromosomal view and define novel features and functions of metazoan programmed DNA elimination.
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•Genome analysis enables a comprehensive chromosome view of nematode DNA elimination•All chromosome ends undergo subtelomeric DNA breaks and de novo telomere addition•All chromosome regions eliminated contain germline expressed genes•Eliminated DNA is sequestered into micronuclei and then cytoplasmic autophagosomes
Comprehensive genome analysis of DNA elimination in the nematode Ascaris by Wang et al. provides a chromosome view of DNA elimination demonstrating all chromosome ends undergo subtelomeric DNA breaks, loss of distal sequences, and de novo telomere healing. The eliminated DNA is incorporated into micronuclei that become cytoplasmic autophagosomes.
Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and ...regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus,
, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat.
encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.
Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune ...cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.
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•NAbs inhibit CHIKV budding by inducing viral glycoprotein coalescence on infected cells•Budding-arrested nucleocapsid-like particles are structurally heterogeneous•NAbs induce clustering of CHIKV replication spherules at sites of budding blockage•Coalesced glycoprotein-NAb complexes activate Fc receptors on effector cells
Jin et al. demonstrate that neutralizing antibodies (NAbs) inhibit chikungunya virus budding by inducing viral glycoprotein coalescence on infected cells. NAbs that crosslink glycoproteins engage Fc receptors on immune cells to activate antiviral responses. Considering the classical entry-inhibition function of NAbs, this work describes a triply functional antiviral pathway for NAbs.
Correct centriole/basal body positioning is required for numerous biological processes, yet how the cell establishes this positioning is poorly understood. Analysis of centriolar/basal body ...duplication provides a key to understanding basal body positioning and function. Chlamydomonas basal bodies contain structural features that enable specific triplet microtubules to be specified. Electron tomography of cultures enriched in mitotic cells allowed us to follow basal body duplication and identify a specific triplet at which duplication occurs. Probasal bodies elongate in prophase, assemble transitional fibers (TF) and are segregated with a mature basal body near the poles of the mitotic spindle. A ring of nine-singlet microtubules is initiated at metaphase, orthogonal to triplet eight. At telophase/cytokinesis, triplet microtubule blades assemble first at the distal end, rather than at the proximal cartwheel. The cartwheel undergoes significant changes in length during duplication, which provides further support for its scaffolding role. The uni1-1 mutant contains short basal bodies with reduced or absent TF and defective transition zones, suggesting that the UNI1 gene product is important for coordinated probasal body elongation and maturation. We suggest that this site-specific basal body duplication ensures the correct positioning of the basal body to generate landmarks for intracellular patterning in the next generation.
Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in ...centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of
, which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.
Multi-ciliary arrays promote fluid flow and cellular motility using the polarized and coordinated beating of hundreds of motile cilia. Tetrahymena basal bodies (BBs) nucleate and position cilia, ...whereby BB-associated striated fibers (SFs) promote BB anchorage and orientation into ciliary rows. Mutants that shorten SFs cause disoriented BBs. In contrast to the cytotaxis model, we show that disoriented BBs with short SFs can regain normal orientation if SF length is restored. In addition, SFs adopt unique lengths by their shrinkage and growth to establish and maintain BB connections and cortical interactions in a ciliary force-dependent mechanism. Tetrahymena SFs comprise at least eight uniquely localizing proteins belonging to the SF-assemblin family. Loss of different proteins that localize to the SF base disrupts either SF steady-state length or ciliary force-induced SF elongation. Thus, the dynamic regulation of SFs promotes BB connections and cortical interactions to organize ciliary arrays.
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