First name
Manuel
Last name
Carminati
Year of Study
Research Center
Thesis Title
Molecular implications of Afadin and Aurora-‐A–mediated phosphorylation of NuMA in spindle orientation
Thesis Abstract
Oriented cell divisions contribute to tissue morphogenesis and homeostasis. Planar
divisions occurring with the spindle within the epithelial plane enlarge sheets and
tubules, while asymmetric cell divisions with the spindle aligned to the apico-basal
polarity axis sustain differentiation programs. Several pathways have been involved in
establishing correct spindle orientation, both in cultured cells and in vivo. Most of
these pathways impinge on the evolutionarily conserved Gαi/LGN/NuMA complexes
that orient the spindle by generating pulling forces on astral microtubules (MTs), via
direct interaction of NuMA with the MT-motors Dynein/Dynactin.
My PhD project focused on the molecular mechanisms underlying the spindle
orientation function of Afadin, and on the relevance of NuMA phosphorylation by
Aurora-A for spindle orientation.
During planar cell divisions, Gαi/LGN/NuMA assemblies are restricted to the lateral
cortex, for molecular reasons that are still unclear. Studies conducted during this
thesis indicate that LGN interacts directly with the junctional and F-actin binding
protein Afadin, and defined the TPR domain of LGN (hereon LGNTPR) and a Cterminal
peptide of Afadin (AfadinPEPT) as the minimal interacting regions retaining
micromolar binding affinity. The crystal structure of the LGNTPR-AfadinPEPT fusion
protein shows that the AfadinPEPT threads along the LGNTPR superhelix with opposite
chain directionality, similarly to what observed for LGN in complex with other
ligands, including NuMA. Consistently, we provided evidence that Afadin competes
with NuMA for binding to LGN. Afadin knock-down in HeLa cells leads to reduced
LGN cortical levels, and unexpectedly also to complete loss of cortical NuMA and
Dynein/Dynactin, and hence spindle misorientation. Importantly, we discovered that
Afadin interacts concomitantly with F-actin and LGN in vitro. Furthermore, we
2
showed that loss of Afadin impairs correct cystogenesis of Caco-2 cells, suggesting
that it plays essential functions in epithelial planar cell divisions. Altogether our data
suggest a model whereby in metaphase Afadin mediates cortical recruitment of
Dynein/Dynactin, by targeting LGN at the lateral cortex via direct and concomitant
interaction with LGN and with cortical F-actin. Later, LGN engages with NuMA and
Dynein/Dynactin to exert pulling forces on the mitotic spindle. Thus, Afadin
represents the first described mechanical anchor between the acto-myosin cell cortex
and Dynein/Dynactin MT-motors.
Besides being spatially regulated, the cortical recruitment of Gαi/LGN/NuMA is
timely controlled by mitotic kinases coordinating spindle orientation with mitotic
progression. It was reported that the activity of the mitotic kinase Aurora-A is
required for correct spindle orientation in human cells in culture, and that NuMA is
among its phosphorylation targets. However, whether NuMA is phosphorylated
directly by Aurora-A and how molecularly its kinase activity affects spindle
orientation was still unknown when we started our studies. Analyses in HeLa and
RPE-1 cells revealed that in metaphase depletion or inhibition of Aurora-A leads to
aberrant accumulation of NuMA at the spindle poles and loss from the cortex, despite
LGN localizes normally at the cortex. FRAP experiments revealed that Aurora-A
governs the dynamic exchange between the cytoplasmic and the spindle polelocalized
pools of NuMA. Experiments in vitro and in cells showed that Aurora-A
phosphorylates directly three serine residues on the C-terminus of NuMA, and
mutation of Ser1969 into alanine recapitulates the aberrant polar accumulation of
NuMA and the spindle orientation defects observed upon Aurora-A inhibition. Thus
we concluded that phosphorylation on Ser1969 of NuMA by Aurora-A controls
3
NuMA distribution between the spindle poles and the overlying cortex, and allows
proper spindle orientation. Intriguingly, Ser1969 lies within a previously
characterized MT-binding domain. In vitro co-sedimentation and bundling assays
revealed that the binding affinity of NuMA for MTs is unaltered by Aurora-Amediated
phosphorylation, suggesting that unphosphorylated NuMA accumulates at
spindle poles via a receptor other than MTs. Most interestingly, with our experiments
we also identified a new MT-binding domain of NuMA positioned downstream of the
LGN binding motif. This result implies that NuMA can simultaneously interact with
LGN and MTs. Based on these findings, we propose that in metaphase the MTbinding
activity of NuMA may contribute to anchor astral MT +TIPs at cortical sites
together with LGN.
divisions occurring with the spindle within the epithelial plane enlarge sheets and
tubules, while asymmetric cell divisions with the spindle aligned to the apico-basal
polarity axis sustain differentiation programs. Several pathways have been involved in
establishing correct spindle orientation, both in cultured cells and in vivo. Most of
these pathways impinge on the evolutionarily conserved Gαi/LGN/NuMA complexes
that orient the spindle by generating pulling forces on astral microtubules (MTs), via
direct interaction of NuMA with the MT-motors Dynein/Dynactin.
My PhD project focused on the molecular mechanisms underlying the spindle
orientation function of Afadin, and on the relevance of NuMA phosphorylation by
Aurora-A for spindle orientation.
During planar cell divisions, Gαi/LGN/NuMA assemblies are restricted to the lateral
cortex, for molecular reasons that are still unclear. Studies conducted during this
thesis indicate that LGN interacts directly with the junctional and F-actin binding
protein Afadin, and defined the TPR domain of LGN (hereon LGNTPR) and a Cterminal
peptide of Afadin (AfadinPEPT) as the minimal interacting regions retaining
micromolar binding affinity. The crystal structure of the LGNTPR-AfadinPEPT fusion
protein shows that the AfadinPEPT threads along the LGNTPR superhelix with opposite
chain directionality, similarly to what observed for LGN in complex with other
ligands, including NuMA. Consistently, we provided evidence that Afadin competes
with NuMA for binding to LGN. Afadin knock-down in HeLa cells leads to reduced
LGN cortical levels, and unexpectedly also to complete loss of cortical NuMA and
Dynein/Dynactin, and hence spindle misorientation. Importantly, we discovered that
Afadin interacts concomitantly with F-actin and LGN in vitro. Furthermore, we
2
showed that loss of Afadin impairs correct cystogenesis of Caco-2 cells, suggesting
that it plays essential functions in epithelial planar cell divisions. Altogether our data
suggest a model whereby in metaphase Afadin mediates cortical recruitment of
Dynein/Dynactin, by targeting LGN at the lateral cortex via direct and concomitant
interaction with LGN and with cortical F-actin. Later, LGN engages with NuMA and
Dynein/Dynactin to exert pulling forces on the mitotic spindle. Thus, Afadin
represents the first described mechanical anchor between the acto-myosin cell cortex
and Dynein/Dynactin MT-motors.
Besides being spatially regulated, the cortical recruitment of Gαi/LGN/NuMA is
timely controlled by mitotic kinases coordinating spindle orientation with mitotic
progression. It was reported that the activity of the mitotic kinase Aurora-A is
required for correct spindle orientation in human cells in culture, and that NuMA is
among its phosphorylation targets. However, whether NuMA is phosphorylated
directly by Aurora-A and how molecularly its kinase activity affects spindle
orientation was still unknown when we started our studies. Analyses in HeLa and
RPE-1 cells revealed that in metaphase depletion or inhibition of Aurora-A leads to
aberrant accumulation of NuMA at the spindle poles and loss from the cortex, despite
LGN localizes normally at the cortex. FRAP experiments revealed that Aurora-A
governs the dynamic exchange between the cytoplasmic and the spindle polelocalized
pools of NuMA. Experiments in vitro and in cells showed that Aurora-A
phosphorylates directly three serine residues on the C-terminus of NuMA, and
mutation of Ser1969 into alanine recapitulates the aberrant polar accumulation of
NuMA and the spindle orientation defects observed upon Aurora-A inhibition. Thus
we concluded that phosphorylation on Ser1969 of NuMA by Aurora-A controls
3
NuMA distribution between the spindle poles and the overlying cortex, and allows
proper spindle orientation. Intriguingly, Ser1969 lies within a previously
characterized MT-binding domain. In vitro co-sedimentation and bundling assays
revealed that the binding affinity of NuMA for MTs is unaltered by Aurora-Amediated
phosphorylation, suggesting that unphosphorylated NuMA accumulates at
spindle poles via a receptor other than MTs. Most interestingly, with our experiments
we also identified a new MT-binding domain of NuMA positioned downstream of the
LGN binding motif. This result implies that NuMA can simultaneously interact with
LGN and MTs. Based on these findings, we propose that in metaphase the MTbinding
activity of NuMA may contribute to anchor astral MT +TIPs at cortical sites
together with LGN.
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