Correct spindle positioning is essential for tissue morphogenesis and homeostasis. The orientation of the mitotic spindle is determined by cortical force generators formed on NuMA:LGN:Gαi complexes, which anchor astral microtubules emanating from the spindle poles at specialized domains of the plasma membrane via direct interaction with the motor proteins Dynein/Dynactin. Cortical polarity cues and actin-associated proteins synergize with extrinsic signals (such as cell-to-cell and cell-to-extracellular matrix contacts) in recruiting NuMA:LGN:Gαi complexes at the cell cortex. In addition, spindle placement is coordinated with mitotic progression by mitotic kinases regulating the timely cortical recruitment of NuMA:LGN:Gαi above the spindle poles. My PhD project focused on the study of the molecular mechanisms accounting for the spindle orientation functions of the Aurora-A kinase, the polarity protein Lgl2, and the junctional protein Afadin. The Aurora-A kinase is known for being implicated in spindle alignment, however the molecular events underlying this function remain to date unclear. To study the spindle orientation functions of Aurora-A, I developed protocols for the partial inhibition of its activity in transformed and non-transformed cells in culture. Under these conditions, in metaphase NuMA and Dynactin accumulate abnormally at the spindle poles without reaching the cortex, while the cortical distribution of LGN remains unperturbed. Fluorescence Recovery After Photobleaching (FRAP) experiments conducted on GFP-NuMA revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle-pole-localized pools of NuMA. Molecularly, Aurora-A phosphorylates directly the C-terminus of NuMA on three serine residues, among which Ser-1969 is the major determinant for the dynamic behaviour of NuMA at the spindle poles. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif, thus suggesting that NuMA can associate concomitantly with LGN and microtubules. This finding indicates that the microtubule-binding activity of NuMA might contribute to anchor microtubule +TIPs at cortical sites with LGN. Collectively, my studies demonstrate that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in cultured cells. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules nor for LGN, but rather determines the mobility of the protein at the spindle-poles. Biochemical studies suggested that Lgl2 can associate with LGN, hinting at a possible role of this protein in spindle orientation in mammalian system. On these premises, I found that depletion of Lgl2 misorients the spindle in HeLa cells plated on fibronectin. However, I could not reproduce the Lgl2:LGN interaction in vitro nor ex vivo. Interestingly, by immunoprecipitation experiments I detected an interaction between NuMA and Lgl2, which could explain the phenotype of spindle misorientation resulting from the silencing of Lgl2 in HeLa cells. Further studies will be required to gain a molecular understanding of the relevance Lgl2:NuMA interaction in oriented divisions. Part of my PhD studies addressed the role of Afadin in spindle orientation; I demonstrated that Afadin is required for spindle positioning, and correct epithelial morphogenesis of Caco-2 three-dimensional cysts. At a molecular level, Afadin binds directly and concomitantly to F-actin and to LGN. Indeed, in mitotic HeLa cells, Afadin is required for cortical accumulation of LGN, NuMA and Dynein above the spindle poles, in a F-actin dependent manner. Collectively, these results uncovered a pivotal role of Afadin in governing the enrichment of LGN and NuMA at the lateral cortex of polarized epithelia. They also depict that Afadin as the first mechanical anchor between Dynein and cortical F-actin.
|Titolo:||MOLECULAR CONTRIBUTION OF THE AURORA-A KINASE AND THE JUNCTIONAL PROTEIN AFADIN TO ORIENTED CELL DIVISIONS|
|Tutor esterno:||MAPELLI, MARINA|
|Data di pubblicazione:||18-mar-2016|
|Parole Chiave:||Spindle orientation; metaphase; planar cell division; NuMA; Afadin; Aurora-A kinase; mitosis; actin; Lgl2; mitotic spindle; MLN8237|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||MOLECULAR CONTRIBUTION OF THE AURORA-A KINASE AND THE JUNCTIONAL PROTEIN AFADIN TO ORIENTED CELL DIVISIONS ; supervisor: M. Mapelli ; internal advisor: G. Scita ; external advisor: C. González. - Milano : Università degli studi di Milano. UNIVERSITA' DEGLI STUDI DI MILANO, 2016 Mar 18. ((27. ciclo, Anno Accademico 2015.|
|Appare nelle tipologie:||Tesi di dottorato|