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STOP proteins

Published on 25 March 2015


Microtubules are fibrous structures in the cytoplasm of eukaryotic cells where they play a vital role in cell morphogenesis organization and motility. In mitotic cells microtubules are centrally involved in the mechanics and control of cell division. Microtubules are key components of the mitotic spindle which is the machinery used by eukaryotic cells to segregate chromosomes during mitosis.

There is strong evidence that abnormalities of elements of the mitotic machinery are major factors favoring genomic instability and tumor progression in human cancers. Microtubules are also important for cytoplasmic organization and organelle trafficking in interphasic cells. These microtubule functions are conspicuous in neurons, whose asymmetry can be extreme with cell extensions at the metric scale.

STOP proteins

Neurons contain abundant subpopulations of stable microtubules that resist depolymerising conditions such as exposure to cold temperature and to depolymerising drugs [Baas et al., 1991 ; Guillaud et al., 1998 ; Job and Margolis, 1984]. In neurons, microtubule stabilization is mainly due to microtubule association with a family of proteins known as STOPs (Stable Tubule Only Polypeptides). STOP proteins were initially characterized as microtubule cold-stabilizing factors whose activity was inhibited by interaction with Ca2+-calmodulin [Job et al., 1981] and subsequent work has shown that STOPs contain bi-functional modules comprised of overlapping calmodulin-binding and microtubule-stabilizing sequences [Bosc et al., 2001]. Neurons contain two major variants of STOP, E-STOP (89 kD) and N-STOP (116 kD). E-STOP is present in mice brain from embryonic stage E16 to adulthood, whereas N-STOP appears at birth and is subsequently expressed in the adult brain [Guillaud et al., 1998; Job and Margolis, 1984 ; Job et al., 1981 ; Bosc et al., 2001 ; Bosc et al., 1996].
Neurons contain abundant subpopulations of stable microtubules that resist depolymerising conditions such as exposure to cold temperature and to depolymerising drugs [Baas et al., 1991 ; Guillaud et al., 1998 ; Job and Margolis, 1984]. In neurons, microtubule stabilization is mainly due to microtubule association with a family of proteins known as STOPs (Stable Tubule Only Polypeptides). STOP proteins were initially characterized as microtubule cold-stabilizing factors whose activity was inhibited by interaction with Ca2+-calmodulin [Job et al., 1981] and subsequent work has shown that STOPs contain bi-functional modules comprised of overlapping calmodulin-binding and microtubule-stabilizing sequences [Bosc et al., 2001]. Neurons contain two major variants of STOP, E-STOP (89 kD) and N-STOP (116 kD). E-STOP is present in mice brain from embryonic stage E16 to adulthood, whereas N-STOP appears at birth and is subsequently expressed in the adult brain [Guillaud et al., 1998; Job and Margolis, 1984 ; Job et al., 1981 ; Bosc et al., 2001 ; Bosc et al., 1996].

STOP deficient mice

STOP function has been investigated in whole animals by studying STOP null mice [Andrieux et al., 2002]. In these mice, microtubule cold stability is suppressed, with no dramatic consequences for mouse organogenesis, viability or brain anatomy. However, STOP -/- mice display multiple synaptic deficits that affect both long- and short-term synaptic plasticity in the hippocampus. These synaptic defects are associated with depleted vesicular pools in glutamatergic nerve terminals and with severe behavioral disorders [Andrieux et al., 2002]. The dopaminergic (DA) status of STOP null mice has been investigated by the M. F. Suaud Chagny team (U512, Lyon) , at both the behavioral and the neurochemical levels. STOP null mice consistently showed a pronounced increase in locomotor activity in basal conditions, following a mild stress or after amphetamine injection. We showed that this hyper locomotor reactivity is associated with an increased DA transmission in the limbic system. The DA transmission alterations involved increased DA efflux evoked by electrical stimulations mimicking physiological stimuli, in the absence of basal abnormalities [Brun et al., 2005]. STOP null mice also exhibit an altered Prepulse inhibition (PPI), alteration observed in schizophrenic patients and thought to reflect a dysfunction of conserved sensori-motor gating mechanisms [Fradley et al., 2005]. Remarkably, neuroleptics treatment specifically leads to a rescue of the Post Tetanic Potentiation (PTP) within glutamatergic neurons, to a partial rescue of the synaptic vesicle pool and to an improvement of the maternal behaviour sufficient to allow pups survival [Andrieux et al., 2002 ; Brun et al., 2005 ; Fradley et al., 2005].

Altogether, these results demonstrate that primary synaptic defects affecting cytoskeletal proteins can cause a combination of neurotransmission disorders that have been expected to occur in human psychosis and more precisely in schizophrenia. In agreement with this hypothesis, the recently reported gene associated with schizophrenia Disrupted-In-Schizophrenia 1 (DISC1) has been described as a multifunctional protein acting on microtubule or microtubule-related components [
Morris et al., 2003]. We have thus proposed that microtubules may represent a novel therapeutic target for anti-psychotic agents. As a test for this hypothesis, we have investigated the ability of one class of microtubule stabilizing drugs, the epothilones, to alleviate null mice behavioral and synaptic defects. Epothilone are anti-mitotic drugs recently develop by pharmaceutical companies for anti cancer therapy [Galsky et al., 2005 ; Kolman 2004 ; Kolman 2004 ; Wang et al., 2005]. We found that epothilone D is able to alleviate STOP KO mice disorders when used 10 to 100 times less than the dosage use in human cancer therapy. Thus, epothilone can act on synaptic plasticity and on animal behavior via its microtubule stabilizing properties. Based on these pharmacological results we patented the use of epothilones for neuronal disorders.

STOP proteins and synapses

STOP KO mice have indicated a probable and intriguing role of STOP at the synapses. Indeed, it has been very surprising that a protein associated with microtubules along the whole neurite turns out to be important for synaptic function, despite apparent microtubule absence in nerve terminals. There is however, biochemical and proteomic evidence that STOP localizes to synapses and this has raised questions concerning the mechanisms that could promote STOP dissociation from microtubules and re-localization in synaptic structures. Long-term plasticity events, which are severely impaired in STOP null mice, are known to involve the calcium/calmodulin-dependent protein kinase II (CaMKII) and we had previous evidence for the presence of potential CaMKII phosphorylation sites on STOP. Thus, we investigate the potential phosphorylation of STOP by CaMKII. We showed that STOP is phosphorylated by CaMKII on at least three independent sites (S139, S198, S491), both in vitro and in vivo and that phosphorylated STOP do not bind to microtubules in vitro. The phosphorylated forms of STOP co-localize with actin-rich structures in cultured neurons and bind to polymerized F-actin in vitro. Thus, STOP phosphorylation by CaMKII may allow STOP association with synaptic actin and be important for synaptic plasticity (PhD thesis of Julie Baratier, 2002-2004, directed by Annie Andrieux. Baratier et al., 2006).

As an effort to understand STOP function at the synapse, we searched for STOP partners using the two-hybrid system. A large screen using several fragments of STOP protein and several cDNA libraries lead to the identification of only two potential partners, Tctex-1 and Arc proteins. TcTex-1 is one of three dynein light chains of the dynein motor complex and has been implicated in targeting and binding cargoes to cytoplasmic dynein for retrograde or apical transport [
Chuang et al., 2001]. Arc, activity-regulated cytoskeleton-associated gene, is an immediate early gene, involved in LTP and other forms of neuroplasticity [Lyford et al., 1995 ; Steward et al., 2001].
In a search for proteins sharing at least one conserved module with STOP we identify of a mammalian protein containing a microtubule-binding domain. In addition, the protein N-terminus is very similar to that of N-STOP and comprises the calmodulin-binding motif Cam1. We have called this protein SL21, for 21 kDa STOP-like protein and we performed molecular and cellular characterization. Surprisingly, in cultured neurons, SL21 mainly associates with the somatic Golgi and with punctuated material in neurites, despite combined in vitro and in vivo evidence that SL21 has calmodulin-binding and microtubule-stabilizing activity, similar to STOPs. We show that the Golgi-targeting sequence of SL21 is located within the N-terminal domain that SL21 shares with STOPs, and that Golgi targeting most probably requires SL21 palmitoylation. We find that deletion mutants of N-STOP, containing the N-terminal domain of N-STOP but lacking microtubule stabilizing modules, are targeted to the Golgi apparatus and that the preferential localization of STOP or of STOP mutants on microtubules depends on the presence of at least two microtubule stabilizing modules in the peptide chain. Recent studies indicate the presence of Golgi material in neurites and in nerve terminals, which may be important for synaptic plasticity. Thus, the STOP protein family, which has capacity to couple Golgi vesicles with the neuronal cytoskeleton, may be important for Golgi trafficking in neurites (Gory-Fauré et al., 2006).

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