The emerging tools for precisely manipulating microtubules.

Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.

CCP5 and CCP6 retain CP110 and negatively regulate ciliogenesis.

BACKGROUND: The axonemal microtubules of primary cilium undergo a conserved protein posttranslational modification (PTM) - polyglutamylation. This reversible procedure is processed by tubulin tyrosine ligase-like polyglutamylases to form secondary polyglutamate side chains, which are metabolized by the 6-member cytosolic carboxypeptidase (CCP) family. Although polyglutamylation modifying enzymes have been linked to ciliary architecture and motility, it was unknown whether they also play a role in ciliogenesis. RESULTS: In this study, we found that CCP5 expression is transiently downregulated upon the initiation of ciliogenesis, but recovered after cilia are formed. Overexpression of CCP5 inhibited ciliogenesis, suggesting that a transient downregulation of CCP5 expression is required for ciliation initiation. Interestingly, the inhibitory effect of CCP5 on ciliogenesis does not rely on its enzyme activity. Among other 3 CCP members tested, only CCP6 can similarly suppress ciliogenesis. Using CoIP-MS analysis, we identified a protein that potentially interacts with CCP - CP110, a known negative regulator of ciliogenesis, whose degradation at the distal end of mother centriole permits cilia assembly. We found that both CCP5 and CCP6 can modulate CP110 level. Particularly, CCP5 interacts with CP110 through its N-terminus. Loss of CCP5 or CCP6 led to the disappearance of CP110 at the mother centriole and abnormally increased ciliation in cycling RPE-1 cells. Co-depletion of CCP5 and CCP6 synergized this abnormal ciliation, suggesting their partially overlapped function in suppressing cilia formation in cycling cells. In contrast, co-depletion of the two enzymes did not further increase the length of cilia, although CCP5 and CCP6 differentially regulate polyglutamate side-chain length of ciliary axoneme and both contribute to limiting cilia length, suggesting that they may share a common pathway in cilia length control. Through inducing the overexpression of CCP5 or CCP6 at different stages of ciliogenesis, we further demonstrated that CCP5 or CCP6 inhibited cilia formation before ciliogenesis, while shortened the length of cilia after cilia formation. CONCLUSION: These findings reveal the dual role of CCP5 and CCP6. In addition to regulating cilia length, they also retain CP110 level to suppress cilia formation in cycling cells, pointing to a novel regulatory mechanism for ciliogenesis mediated by demodifying enzymes of a conserved ciliary PTM, polyglutamylation.

NAP1L4 inhibits porcine circovirus type 2 replication via IFN-ß signaling pathway.

Porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to interact with nucleosome assembly protein 1-like 4 (NAP1L4). The biological function of Cap-NAP1L4 interaction is unknown. Here, we demonstrated that PCV2 Cap could directly interact with NAP1L4, which the amino acid residues 124-279 of NAP1L4 were responsible for the interaction. Furthermore, over-expression of NAP1L4 down-regulated the expression of PCV2 Cap and Rep. DNA copies and virus titers were also decreased in NAP1L4 over-expressed PK15 cells. While, knockout of NAP1L4 through CRISPR/Cas9 technology in PK15 cells could up-regulate the mRNA and protein levels of PCV2 Cap and Rep. PCV2 genomic DNA copies and virus titers were also increased in NAP1L4-knockdown/-knockout PK15 cells compared with wild type PK15 cells. In addition, NAP1L4 depletion was demonstrated to facilitate cytosolic carboxypeptidase-like protein 5 (CCP5) and cytosolic carboxypeptidase 6 (CCP6) expression, which could activate cGAS to promote IFN-ß production. Indeed, knockout of NAP1L4 was also confirmed to increase IFN-ß expression. And IFN-ß stimulation could promote PCV2 replication in PK15 cells. Taken together, our findings suggest that NAP1L4 interacts with PCV2 Cap and inhibits PCV2 replication through regulating IFN-ß production. Our study provides theoretical basis for further study of PCV2.

Toxoplasma gondii oocyst-driven infection in pigs, chickens and humans in northeastern China.

BACKGROUND: Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect almost all warm-blooded animals. The aim of the present study was to investigate T. gondii oocyst-driven infection in pigs, chickens and humans in Jilin province, northeastern China. RESULTS: The serum samples of pigs, chickens and humans were sampled and tested by indirect enzyme-linked immunosorbent assays (ELISAs) using dense granule antigen GRA7, oocyst-specific protein OWP8, and sporozoite-specific protein CCp5A, respectively. Results showed a prevalence of 16.7% by GRA7-ELISA, and 12.2% by OWP8- and CCp5A-ELISA in pigs; 10.4% by GRA7-ELISA, 13.5% by OWP8-ELISA, and 9.4% by CCp5A-ELISA in chickens; and 14.2% by GRA7-ELISA, 3.6% by OWP8-ELISA, and 3.0% by CCp5A-ELISA in humans. No significant differences were observed between T. gondii seroprevalence in pigs and chickens among the three antigens-based ELISAs (P > 0.05). However, there were significant differences between T. gondii seroprevalence rates in humans (P < 0.05). These findings demonstrated a low prevalence of T. gondii oocyst-driven infection in humans, a medium prevalence in pigs, and a high prevalence in chickens. CONCLUSIONS: The present study demonstrated that different oocyst-driven infection rates in different animal species, which would help to design effective strategies to prevent T. gondii transmission. To our knowledge, this is the first study to differentiate T. gondii infective forms in pigs, chickens and humans in China.