Human Cytomegalovirus Nuclear Egress Complex Subunit, UL53, Associates with Capsids and Myosin Va, but Is Not Important for Capsid Localization towards the Nuclear Periphery.

After herpesviruses encapsidate their genomes in replication compartments (RCs) within the nuclear interior, capsids migrate to the inner nuclear membrane (INM) for nuclear egress. For human cytomegalovirus (HCMV), capsid migration depends at least in part on nuclear myosin Va. It has been reported for certain herpesviruses that the nucleoplasmic subunit of the viral nuclear egress complex (NEC) is important for this migration. To address whether this is true for HCMV, we used mass spectrometry and multiple other methods to investigate associations among the HCMV NEC nucleoplasmic subunit, UL53, myosin Va, major capsid protein, and/or capsids. We also generated complementing cells to derive and test HCMV mutants null for UL53 or the INM NEC subunit, UL50, for their importance for these associations and, using electron microscopy, for intranuclear distribution of capsids. We found modest associations among the proteins tested, which were enhanced in the absence of UL50. However, we found no role for UL53 in the interactions of myosin Va with capsids or the percentage of capsids outside RC-like inclusions in the nucleus. Thus, UL53 associates somewhat with myosin Va and capsids, but, contrary to reports regarding its homologs in other herpesviruses, is not important for migration of capsids towards the INM.

Integrated proteogenetic analysis reveals the landscape of a mitochondrial-autophagosome synapse during PARK2-dependent mitophagy.

The PINK1 protein kinase activates the PARK2 ubiquitin ligase to promote mitochondrial ubiquitylation and recruitment of ubiquitin-binding mitophagy receptors typified by OPTN and TAX1BP1. Here, we combine proximity biotinylation of OPTN and TAX1BP1 with CRISPR-Cas9-based screens for mitophagic flux to develop a spatial proteogenetic map of PARK2-dependent mitophagy. Proximity labeling of OPTN allowed visualization of a "mitochondrial-autophagosome synapse" upon mitochondrial depolarization. Proximity proteomics of OPTN and TAX1BP1 revealed numerous proteins at the synapse, including both PARK2 substrates and autophagy components. Parallel mitophagic flux screens identified proteins with roles in autophagy, vesicle formation and fusion, as well as PARK2 targets, many of which were also identified via proximity proteomics. One protein identified in both approaches, HK2, promotes assembly of a high-molecular weight complex of PINK1 and phosphorylation of ubiquitin in response to mitochondrial damage. This work provides a resource for understanding the spatial and molecular landscape of PARK2-dependent mitophagy.

A human organoid system that self-organizes to recapitulate growth and differentiation of a benign mammary tumor.

As 3D culture has become central to investigation of tissue biology, mammary epithelial organoids have emerged as powerful tools for investigation of epithelial cell polarization and carcinogenesis. However, most current protocols start from single cells suspended in Matrigel, which can also restrict cell differentiation and behavior. Here, we show that the noncancerous mammary cell line HMT-3522 S1, when allowed to spontaneously form cell aggregates ("spheroids") in medium without Matrigel, switches to a collective growth mode that recapitulates many attributes of "usual ductal hyperplasia" (UDH), a common benign mammary lesion. Interestingly, these spheroids undergo a complex maturation process reminiscent of embryonic development: solid-cell cords form their own basement membrane, grow on the surface of initially homogeneous cell aggregates, and form asymmetric lumina lined by two distinct cell types that express basal and luminal cytokeratins. This sequence of events provides a cellular mechanism that explains how the characteristic crescent-shaped, asymmetrical lumina form in UDH. Our results suggest that HMT-3522 S1 spheroids are useful as an in vitro model system to study UDH biology, glandular lumen formation, and stem cell biology of the mammary gland.

Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol.

Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B-containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.

Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis.

To achieve mitosis and cytokinesis, microtubules must assemble into distinct structures at different stages of cell division-mitotic spindles to segregate the chromosomes before anaphase and midzones to keep sister genomes apart and guide the cleavage furrow after anaphase. This temporal regulation is believed to involve Cdk1 kinase, which is inactivated in a switch-like way after anaphase. We found that inhibiting Plk1 caused premature assembly of midzones in cells still in metaphase, breaking the temporal regulation of microtubules. The antiparallel microtubule-bundling protein PRC1 plays a key role in organizing the midzone complex. We found that Plk1 negatively regulates PRC1 through phosphorylation of a single site, Thr-602, near the C-terminus of PRC1. We also found that microtubules stimulated Thr-602 phosphorylation by Plk1. This creates a potential negative feedback loop controlling PRC1 activity. It also made the extent of Thr-602 phosphorylation during mitotic arrest dependent on the mechanism of the arresting drug. Unexpectedly, we could not detect a preanaphase regulatory role for Cdk1 sites on PRC1. We suggest that PRC1 is regulated by Plk1, rather than Cdk1 as previously proposed, because its activity must be spatiotemporally regulated both preanaphase and postanaphase, and Cdk1 activity is too binary for this purpose.

Functional analysis of VopF activity required for colonization in Vibrio cholerae.

Vibrio cholerae, a Gram-negative facultative pathogen, is the etiologic agent for the diarrheal disease cholera. We previously characterized a clinical isolate, AM-19226, that translocates a type III secretion system (T3SS) effector protein with actin-nucleating activity, VopF, into the host cells. From comparative genomic studies, we identified a divergent T3SS island in additional isolates which possess a VopF homolog, VopN. Unlike the VopF-mediated protrusion formation, VopN localizes to stress fiber in host cells similarly to VopL, which is present in the pandemic strain of Vibrio parahaemolyticus. Chimera and yeast two-hybrid studies indicated that the amino-terminal regions of VopF and VopN proteins interact with distinct host cell factors. We determined that AM-19226-infected cells are arrested at S phase of the cell cycle and that VopF/VopN are antiapoptotic factors. To understand how VopF may contribute to the pathogenesis of AM-19226, we examined the effect of VopF in an in vitro polarized-epithelial model and an in vivo adult rabbit diarrheal model. Within the T3SS pathogenicity island is VopE, a homolog of YopE from Yersinia, which has been shown to loosen tight junctions. In polarized intestinal epithelia, VopF and VopE compromised the integrity of tight junctions by inducing cortical actin depolymerization and aberrant localization of the tight-junction protein ZO-1. An assay for pathogenicity in the adult rabbit diarrhea model suggested that these effectors are involved in eliciting the diarrheal response in infected rabbits.

Interaction between Poly(ADP-ribose) and NuMA contributes to mitotic spindle pole assembly.

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function.

Poly(ADP-ribose) (PAR) is a large, negatively charged post-translational modification that is produced by polymerization of NAD+ by PAR polymerases (PARPs). There are at least 18 PARPs in the human genome, several of which have functions that are unknown. PAR modifications are dynamic; PAR structure depends on the balance between synthesis and hydrolysis by PAR glycohydrolase2. We previously found that PAR is enriched in vertebrate somatic-cell mitotic spindles and demonstrated a requirement for PAR in the assembly of Xenopus egg extract spindles. Here, we knockdown all characterized PARPs using RNA interference (RNAi), and identify tankyrase-1 as the PARP that is required for mitosis. Tankyrase-1 localizes to mitotic spindle poles, to telomeres and to the Golgi apparatus. Tankyrase-1 RNAi was recently shown to result in mitotic arrest, with abnormal chromosome distributions and spindle morphology observed--data that is interpreted as evidence of post-anaphase arrest induced by failure of telomere separation6. We show that tankyrase-1 RNAi results in pre-anaphase arrest, with intact sister-chromatid cohesion. We also demonstrate a requirement for tankyrase-1 in the assembly of bipolar spindles, and identify the spindle-pole protein NuMA as a substrate for covalent modification by tankyrase-1.

XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly.

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.