As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In C57BL/6 male mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant-delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre-and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdyn Cre neurons using Channelrhodopsin-2 disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence.

At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT—enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs)—phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.

Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre- and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdynCre neurons using Channelrhodopsin-2 (ChR2) disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence.

20Mitotic centrosomes are complex membraneless organelles that guide the formation of 21 mitotic spindles to ensure faithful cell division. They are formed by timely expansion of the 22 pericentriolar material (PCM) around the centrioles at the onset of mitosis. How PCM proteins 23 are recruited and held together without a lipid membrane remains elusive. Here we found that 24 endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses 25 into liquid-like granules during early mitosis in cultured human cells. Furthermore, the N-terminal 26 segment of PCNT, enriched with conserved coiled-coils and low-complexity regions (LCRs), 27 undergoes phase separation. These PCNT "condensates" selectively recruit PCM components 28 and nucleate microtubules in cells. We propose that coiled-coils and LCRs, two prevalent 29 sequence features in the centrosomal proteome, are preserved under evolutionary pressure to 30 drive phase separation, a process that bestows upon the centrosome a distinct material 31 property critical for its assembly and functions. 32 128 supplement 3). This dynamic behavior suggests that these PCNT granules may be liquid-like 129 condensates formed by liquid-liquid phase separation. To test this hypothesis, we treated the 130 cells with aliphatic alcohol 1,6-hexanediol, which dissolves liquid-like condensates by disrupting 131 weak hydrophobic interactions (Kroschwald et al., 2017; Ribbeck & Gorlich, 2002). Following 132 hexanediol addition, PCNT granules near centrosomes dispersed within minutes (Figure 1B133 and C). Together, these results suggest that PCNT, expressed from its endogenous locus, 134 8 forms liquid-like condensates, likely through liquid-liquid phase separation, before incorporating 135 into the early mitotic centrosome of cultured human cells. 136 137 Coiled-coils and low-complexity regions of pericentrin are more conserved than the rest 138 of the protein sequence 139 157 2-figure supplement 1). However, as a known limitation with current disorder predictions 158 9 ( Atkins et al., 2015), not all disorder predictors are in complete agreement, with each predictor 159 suggesting different degrees of disorder/order tendency [e.g., IUpred (Dosztanyi et al., 2005) 160 predicted an overall more ordered structure than PONDR did]. 161Nonetheless, by comparing the conservation profile of the multi-species alignment and the 162 locations of the predicted features of human PCNT, we found that the coiled-coils and LCRs in 163 human PCNT are concentrated within the conserved orthologous regions (Figure 2A). 164Statistical analyses further demonstrated that coiled-coils and LCRs are significantly more 165 conserved than non-coiled-coils and non-LCRs, respectively, in human PCNT (Figure 2B). 166Together, these results suggest that coiled-coils and LCRs of pericentrin orthologs are likely 167 under natural selection to preserve their molecular functions. 168 169 PCNT phase separates via its coiled-coil/LCR-rich segments in a concentration-170 dependent manner 171 Given tha...