Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology

Stabilization of Gaze during Early Xenopus Development by Swimming-Related Utricular Signals.

International audienceLocomotor maturation requires concurrent gaze stabilization improvement for maintaining visual acuity [1, 2]. The capacity to stabilize gaze, in particular in small aquatic vertebrates where coordinated locomotor activity appears very early, is determined by assembly and functional maturation of inner ear structures and associated sensory-motor circuitries [3-7]. Whereas utriculo-ocular reflexes become functional immediately after hatching [8, 9], semicircular canal-dependent vestibulo-ocular reflexes (VOR) appear later [10]. Thus, small semicircular canals are unable to detect swimming-related head oscillations, despite the fact that corresponding acceleration components are well-suited to trigger an angular VOR [11]. This leaves the utricle as sole vestibular origin for swimming-related compensatory eye movements [12, 13]. We report a remarkable ontogenetic plasticity of swimming-related head kinematics and vestibular endorgan recruitment in Xenopus tadpoles with beneficial consequences for gaze-stabilization. Swimming of older larvae generates sinusoidal head undulations with small, similar curvature angles on the left and right side that optimally activate horizontal semicircular canals. Young larvae swimming causes left-right head undulations with narrow curvatures and strong, bilaterally dissimilar centripetal acceleration components well-suited to activate utricular hair cells and to substitute the absent semicircular canal function at this stage. The capacity of utricular signals to supplant semicircular canal function was confirmed by recordings of eye movements and extraocular motoneurons during off-center rotations in control and semicircular canal-deficient tadpoles. Strong alternating curvature angles and thus linear acceleration profiles during swimming in young larvae therefore represents a technically elegant solution to compensate for the incapacity of small semicircular canals to detect angular acceleration components

Swim style-dependent utricular activation ensures gaze-stabilization during early development

International audienceLocomotor maturation requires concurrent gaze stabilization improvement for maintaining visual acuity [1, 2]. The capacity to stabilize gaze, in particular in small aquatic vertebrates where coordinated locomotor activity appears very early, is determined by assembly and functional maturation of inner ear structures and associated sensory-motor circuitries [3-7]. Whereas utriculo-ocular reflexes become functional immediately after hatching [8, 9], semicircular canal-dependent vestibulo-ocular reflexes (VOR) appear later [10]. Thus, small semicircular canals are unable to detect swimming-related head oscillations, despite the fact that corresponding acceleration components are well-suited to trigger an angular VOR [11]. This leaves the utricle as sole vestibular origin for swimming-related compensatory eye movements [12, 13]. We report a remarkable ontogenetic plasticity of swimming-related head kinematics and vestibular endorgan recruitment in Xenopus tadpoles with beneficial consequences for gaze-stabilization. Swimming of older larvae generates sinusoidal head undulations with small, similar curvature angles on the left and right side that optimally activate horizontal semicircular canals. Young larvae swimming causes left-right head undulations with narrow curvatures and strong, bilaterally dissimilar centripetal acceleration components well-suited to activate utricular hair cells and to substitute the absent semicircular canal function at this stage. The capacity of utricular signals to supplant semicircular canal function was confirmed by recordings of eye movements and extraocular motoneurons during off-center rotations in control and semicircular canal-deficient tadpoles. Strong alternating curvature angles and thus linear acceleration profiles during swimming in young larvae therefore represents a technically elegant solution to compensate for the incapacity of small semicircular canals to detect angular acceleration components

Morphological changes induced by Bmcc1s overexpression requires MAP6.

<p>Confocal microscopy image projections of cells transfected with a Bmcc1s-V5 or GFP expressing plasmid, and stained for V5 (green) and F-actin (detected with TRITC-conjugated phalloidin in red). Cells were fixed 24 h after transfection. (A) primary astrocytes; (B) primary neurons. The morphology of GFP-expressing cells (green) was unchanged compared to untransfected cells. In contrast, Bmcc1s-V5-expressing astrocytes and neurons developed numerous membrane protrusions (white arrowheads). Images in B illustrate representative confocal projections of the effect of Bmcc1s-V5 on neuritic growth and number in wild-type neurons. The whole Bmcc1s-V5 transfected neuron is shown in the insert. Histograms present means ± sd of the length of the longest neurite and of the number of neurites. *** p-value<0.0001 ** p-value<0.001. ns, not significant for 3 independent experiments using the two sample independent t-test. In neurons, length of the longest neurite, and number of neurites (or cell extensions starting from the soma) were significantly increased by Bmcc1s-V5 transfection, but not in MAP6-deleted neurons. Bars: 10 µm.</p

Subcellular localization of Bmcc1s in primary neurons.

<p>(A–C) Confocal section images of primary neurons after 7 days of culture immunostained for endogenous Bmcc1s (green) and α-tubulin or neurofilament subunit M (NF-M) (red). Merge images showed that Bmcc1s colocalizes with α-tubulin (A) and NF-M (C) immunoreactivity signal. Boxed regions in A and C indicate the fields enlarged in each image. B. In nocodazole-treated primary neurons (10 µM, 1 h), Bmcc1s followed the disrupted α-tubulin microtubular staining. (D) Immunogold labeling and electron microscopy analysis of primary neurons showed that Bmcc1s localized on cytoskeleton-type structures compatible with microtubules (left) and intermediate filaments (right). Bars: 10 µm (A–C); 100 nm (D).</p

Immunodetection of Bmcc1s.

<p>(A) Immunoblot of Bmcc1s in lysates of HeLa cells transfected with a plasmid expressing Bmcc1s-V5. Similar profiles were obtained using the Bmcc1 antiserum or anti-V5 antibodies. Note that the Bmcc1 antiserum recognized an endogenous protein around 50 kDa (arrow) of the same size as Bmcc1s in untransfected HeLa cells. Immunostaining of HeLa cells transfected with a plasmid expressing Bmcc1s-V5, using either the Bmcc1 antiserum or anti-V5 antibodies. The antiserum detected only the V5 positive cells, and both signals overlapped. Scale bar: 100 µm (B) Immunoblot of endogenous Bmcc1 isoforms in mouse tissue lysates using Bmcc1 antiserum. GAPDH expression is shown as a loading reference. As in HeLa cells expressing Bmcc1s-V5, the Bmcc1 antiserum detected a band around 50 kDa (arrow) in the brain lysate that appeared specific to this tissue and was the most abundant among the Bmcc1 isoforms. (C) Immunoblot of endogenous Bmcc1 in primary cultures of astrocyte and neuron lysates at DIV7, using Bmcc1 antiserum. As found in brain tissues, a major band around 50 kDa was detected (arrow).</p

Microtubule Co-sedimentation assays.

<p>Taxol stabilized microtubules were incubated with or without GST-Bmcc1s. The samples were then sedimented through a 60% glycerol cushion. The supernatants (S) and pellets (P) were separated by SDS-PAGE and stained with Coomassie Blue. Tubulin (50 kDa) was mostly present in the pellet fraction with or without Bmcc1s, while Bmcc1s was only detectable in the supernatant.</p

Subcellular localization of Bmcc1s in primary cultures of astrocyte.

<p>(A–C) Confocal section images of primary astrocytes immunostained for endogenous Bmcc1s (green) and α-tubulin or GFAP (red). Merge images showed that Bmcc1s forms punctate spots mainly distributed along α-tubulin stained microtubules (A) and partially colocalized with GFAP-positive intermediate filaments (C). Boxed regions in A indicate the fields enlarged in each image. B. In nocodazole-treated primary astrocytes (10 µM, 1 h), Bmcc1s followed the disrupted α-tubulin microtubular staining. (D) Immunogold labelling and electron mircroscopy analysis of primary astrocytes showed that Bmcc1s localized on cytoskeleton-type structures compatible with microtubules (left) and intermediate filaments (right). Bars: 10 µm (A–C); 200 nm (D).</p

Structure of Bmcc1s.

<p>(A) Schematic representation of mouse Bmcc1 gene. Exons are boxed, in black for the coding sequence and in white for the 5′ and 3′ non-coding sequences. Primers for 5′ RACE and RTPCR experiments are indicated by arrows under exons 11, 12 and 21. (B) Schematic representation of mouse Bmcc1 transcript. (C) Schematic representation of Bmcc1s cDNA and protein. The BNIP2 homology and BCH domains are indicated. Asterisks show the antigenic peptides used to generate the Bmcc1s antiserum.</p