Functional characterization of the novel centrosomal protein Nlp (ninein-like protein)

The centrosome is the major microtubule organizing centre (MTOC) in
animal cells. Most microtubules (MTs) emanate from the centrosome,
where gamma-tubulin ring complexes (gammaTuRCs) act as templates
for MT nucleation. During interphase, the centrosome organizes a MT
array that imparts shape and polarity to the cell and is essential
for intracellular transport and positioning of organelles such as
the Golgi apparatus. During mitosis, centrosomes ensure bipolarity
and correct orientation of the spindle by forming the spindle
poles. In order to switch from the interphasic to the mitotic
state, the centrosome undergoes a structural reorganization, termed
maturation, which is mainly characterized by an increase in MT
nucleation activity. A full appreciation of how centrosomes
contribute to cellular functions requires the isolation and
characterization of unknown centrosome-associated molecules. Here
we describe the identification and characterization of a novel
centrosomal component, the human protein Nlp (ninein-like protein)
related to the previously characterized MT-anchoring protein
ninein. In the first part of the present thesis we describe the
identification of Nlp as a novel centrosomal substrate of Polo-like
kinase 1 (Plk1), an important regulator of mitosis whose activity
is required for centrosome maturation. Nlp interacts with two
distinct gammaTuRC components, gamma-tubulin and hGCP4, and
stimulates MT nucleation. Plk1 phosphorylates Nlp and disrupts its
centrosomal association. Overexpression of an Nlp mutant lacking
Plk1 phosphorylation sites induces defects in mitotic spindle
formation. We propose that Nlp acts as a gammaTuRC binding protein
(GTBP), contributing to the MT nucleation activity of the
centrosome during interphase. At the onset of mitosis, the
displacement of Nlp from the centrosome triggered by Plk1
phosphorylation could represent an important step in the maturation
process which allows the centrosome to switch from the interphasic
to the mitotic state. Thus, we conclude that Nlp, as well as the
related protein ninein, plays an important role in MT organization.
However the function of these two proteins possibly diverged during
evolution: whilst Nlp gained a more prominent role in MT
nucleation, ninein became principally involved in MT anchoring. In
the second part of this thesis we report the initial
characterization of the molecular mechanisms underlying the ability
of Nlp and ninein to induce the fragmentation of the Golgi
apparatus when overexpressed in human cells. We show that the
ability of these two centrosomal proteins to affect the
organization of the Golgi clearly depends on their capacity to
associate with the cytoplasmic dynein-dynactin complex, a molecular
motor complex primarly involved in the maintainance of Golgi
architecture. We propose that the excess of Nlp and ninein could
induce the disruption of the Golgi apparatus by sequestering the
dynein-dynactin complexes. Future investigations should be aimed at
understanding whether the dissociation of the Golgi apparatus from
the centrosome induced by the excess of Nlp and ninein could
interfere with cell migration and cell polarization processes,
which require a highly coordinated action of these two organelles.
Cell migration and cell polarization represent critical events for
immune responses as well as for embryonic development, invasive
growth and metastasis. Thus, our findings raise the interesting
possibility that an upregulation in the expression levels of
structural centrosomal proteins could represent the molecular basis
for developmental disorders and malfunctioning of the immune system
and, on the other hand, modulate the acquisition of invasive
properties by neoplastic cells.