Analysis of role of AKTIP at different stages of cell division

AKTIP is a recently discovered non-shelterin telomeric protein, whose deficiency generates telomere fragility by impinging on replication (Burla et al., 2015). As previusly described, AKTIP interacts with lamins and its downregulation triggers a premature aging phenotype both at cellular level and at organismal level (in a mouse model knocked down for its murine homologue Ft1) recalling those of progeroid patients with mutations in LMNA gene (Burla et al., 2016; La Torre et. al., 2018). Another AKTIP peculiarity is its intracellular distribution: in interphases cells AKTIP is located, as discrete foci, both in the cytoplasm and in the nucleus, where it is especially enriched at nuclear envelope. In mitosis AKTIP is located at spindle matrix in metaphase and then it is enriched in anaphase at the bridge structure connecting the two newly formed cells, called midbody (Burla et al., 2015; 2016). Given these premises, this work aims at thouroughly investigating the AKTIP role and distribution at these two important cellular compartments. Concerning AKTIP at nuclear periphery we focused our attention on its relationship with lamins both in physiological and pathological conditions. Using Structure Illumination Microscopy (SIM) (Schermelleh et al., 2010) we demonstrated that AKTIP is localized at nuclear rim in a close proximity with A-type lamins and that it is mislocated in cells from LMNA-mutations associated progeroid patients suggesting that this mislocalization at nuclear rim could contribute to the HGPS phenotype. For AKTIP at midbody we described by SIM that AKTIP forms a super-molecular structure, with a shape of a ring, around microtubules at the center of the intercellular bridge where the endosomal sorting complex required for transport (ESCRT) complex is recruited and acts to finalize abscission (Elia et al., 2011; Karasmanis et al., 2019). ESCRTs are protein complexes involved in membrane remodeling acting through their highly regulated sequential recruitment at target membrane in collaboration with associated proteins (Gatta and Carlton, 2019). We also showed that AKTIP ring is in spatial proximity with ESCRT-III subunits, and functionally, AKTIP reduction impinges on ESCRT-III IST1 recruitment at the midbody and causes abscission defects, including longer abscission time and multinucleation. The collected data and AKTIP intracellular distribution suggest that AKTIP could act as an ESCRT protein. This hypothesis is further supported by the structural and bioinformatics similarity with TSG101 and by the identification of putative partners belonging to different ESCRTs involving pathways through a proteins interactions screening. We found that AKTIP interactes with an ESCRT-I member, the VPS28 subunit. Since VPS28 bridges the ESCRT-I to the ESCRT-II complex (Carlton and Martin-Serrano; 2007), these data make hypothesize a sequential pathway in which AKTIP is connected to ESCRT-II and then to ESCRT-III via VPS28. In summary, these data taken together indicate AKTIP as a new candidate factor associated with the ESCRT complex functioning at nuclear envelope and in cytokinesis.