Microtubule plus-end tracking proteins (+TIPs) are structurally and functionally diverse factors that accumulate at the growing microtubule plus-ends, connect them to various cellular structures, and ...control microtubule dynamics 1, 2. EB1 and its homologs are +TIPs that can autonomously recognize growing microtubule ends and recruit to them a variety of other proteins. Numerous +TIPs bind to end binding (EB) proteins through natively unstructured basic and serine-rich polypeptide regions containing a core SxIP motif (serine-any amino acid-isoleucine-proline) 3. The SxIP consensus sequence is short, and the surrounding sequences show high variability, raising the possibility that undiscovered SxIP containing +TIPs are encoded in mammalian genomes. Here, we performed a proteome-wide search for mammalian SxIP-containing +TIPs by combining biochemical and bioinformatics approaches. We have identified a set of previously uncharacterized EB partners that have the capacity to accumulate at the growing microtubule ends, including protein kinases, a small GTPase, centriole-, membrane-, and actin-associated proteins. We show that one of the newly identified +TIPs, CEP104, interacts with CP110 and CEP97 at the centriole and is required for ciliogenesis. Our study reveals the complexity of the mammalian +TIP interactome and provides a basis for investigating the molecular crosstalk between microtubule ends and other cellular structures.
► Combining proteomics with bioinformatics is an effective way of +TIP identification ► SxIP motif-containing EB-binding +TIPs are numerous in mammalian genomes ► CEP104 is an EB-binding +TIP involved in ciliogenesis ► AMER2 reveals microtubule tip-plasma membrane contacts
Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ...ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.
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•PLK4 activity is needed in G1 for centriole biogenesis•PLK4 binds its substrate STIL upon inactivation of CDK1-CyclinB•CDK1-CyclinB binds to STIL in mitosis through the same domain to which PLK4 binds•CDK1-CyclinB prevents PLK4 from binding and phosphorylating STIL
Zitouni, Francia et al. explore the mechanisms coupling the cell cycle to centrosome biogenesis, and show that in mitosis, CDK1 competes with PLK4, the trigger of centriole biogenesis, for binding to its substrate STIL. PLK4 binding and phosphorylation of STIL occurs only upon CDK1 activity drop at mitotic exit, leading to centriole biogenesis onset.
Centrioles are very small microtubule-based organelles essential for centrosome, cilia and flagella assembly, which are involved in a variety of cellular and developmental processes. Although the ...centriole was first described almost a century ago, the knowledge on its assembly mechanism remains poor. In the past decade, forefront functional studies have provided important data on the different players involved in centriole biogenesis. Centriole research has now started to profit from highly sensitive structural, imaging, and biochemical techniques that are unveiling how those players contribute to assemble such a small and complex structure. We will review those studies and discuss how this field will increasingly benefit from the newborn and exciting era of super resolution analyses.
Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and ...interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general “microtubule tip localization signal” (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
The kinesin-13 family member mitotic centromere-associated kinesin (MCAK) is a potent microtubule depolymerase
1–4. Paradoxically, in cells it accumulates at the growing, rather than the shortening, ...microtubule plus ends. This plus-end tracking behavior requires the interaction between MCAK and members of the end-binding protein (EB) family
5–8, but the effect of EBs on the microtubule-destabilizing activity of MCAK and the functional significance of MCAK accumulation at the growing microtubule tips have so far remained elusive. Here, we dissect the functional interplay between MCAK and EB3 by reconstituting EB3-dependent MCAK activity on dynamic microtubules in vitro. Whereas MCAK alone efficiently blocks microtubule assembly, the addition of EB3 restores robust microtubule growth, an effect that is not dependent on the binding of MCAK to EB3. At the same time, EB3 targets MCAK to growing microtubule ends by increasing its association rate with microtubule tips, a process that requires direct interaction between the two proteins. This EB3-dependent microtubule plus-end accumulation does not affect the velocity of microtubule growth or shortening but enhances the capacity of MCAK to induce catastrophes. The combination of MCAK and EB3 thus promotes rapid switching between microtubule growth and shortening, which can be important for remodeling of the microtubule cytoskeleton.
► EB3 recruits MCAK to growing microtubule plus ends in vitro ► EB3 protects growing microtubule plus ends from the destabilizing activity of MCAK ► EB3 promotes MCAK-mediated catastrophes by concentrating MCAK at microtubule tips ► The combination of MCAK and EB3 makes microtubules highly dynamic
End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule ...dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.
Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and ...manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed.
The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), ...which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or
How centrosomes form
is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of
egg extracts, where PLK4 can induce
MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that
, PLK4 can self-assemble into condensates that recruit α- and β-tubulins. In
extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs
The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.
The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we ...identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.
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