The Homeobox Transcription Factor Barx2 Regulates Plasticity of Young Primary Myofibers

Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool.We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2.We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation

Extensive Fusion of Mitochondria in Spinal Cord Motor Neurons

<div><p>The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.</p> </div

Constitutive redistribution of labeled mitochondria.

<p>A) Images of a living motor neuron (24 div) were taken within a minute after photo-converting the mitoKaede protein, and then after 18 hours. Over a period of 18 hours mitochondria from the periphery appeared to extensively fuse with organelles from the cell body. Blue circles in the leftmost panels delineate the area of the neuron that was exposed to blue light. Relative fluorescence intensity levels are directly comparable. Scale bars correspond to 20 microns. B) Quantitation of the redistribution of red and green fluorescent mitoKaede. The mean fluorescence intensity per pixel was calculated for both red and green pixels in masks generated solely based on the distribution of red pixels. For each cell (n = 33), the ratio of red: green mean pixel intensity at time 0 h and after 18 h (19±1 h) is plotted as a box and whisker plot. A decrease in this ratio reflects a dilution of the finite amount of red fluorescent mitoKaede with green fluorescent mitoKaede. The median ratio changed from 4.9 to 0.17. C) The level of expression of a short-lived GFP in motor neurons before and after treatment with CHI was quantified by manually generating masks that encompass the green pixels in the cell soma, and calculating the mean fluorescence intensity (n = 10; p<0.001). D) Patch clamp recordings were performed on infected spinal cord neurons sampled from 4 coverslips (n = 7). Spontaneous spiking activity was recorded over 20 s, and action potentials were blocked in all 7 cases by 1 µM TTX. Data are plotted as mean firing rate of the same cells before and after addition of TTX to the ACSF (p<0.001). E) Pooled data showing no significant effect of cycloheximide (CHI 10 µg per ml, n = 20) or tetrodotoxin (TTX, 1 µM, n = 16) on the redistribution of labeled mitochondria over time compared to untreated motor neurons. For each cell in each treatment group, the ratio of red: green mean pixel intensity after 18 h (19±1 h) is normalized to the starting ratio in the same cell, and plotted as a box and whisker plot. F) Box and whisker plots of the change in the size of masks generated from the distribution of red pixels reflecting the dispersion of red fluorescent mitoKaede over time.</p

Inhibition of neuronal activity with TTX does not affect mitochondrial fusion in neurites.

<p>Kymographs show that fusion of mitochondria was unaffected by continuously blocking neuronal activity with TTX (1 µM final concentration). Time lapse images were taken every 10 seconds for 30 minutes at 0 h, 4 h, and 18 h following the marking of a subpopulation of mitochondria within a living neurite (27 div).</p

Time course of the fusion of individual mitochondria.

<p>A living neurite (28 div) with mitochondria containing photoconverted mitoKaede was imaged over several hours. The appearance of green fluorescing mitoKaede within individual red fluorescing mitochondria (labeled i- iii) during 4 hours of observation is seen. The arrow indicates the location of a mitochondrion that had entered the field of view before the first image was taken. By 20 hours, the red fluorescent protein was distributed throughout the mitochondria under observation. Scale bars correspond to 5 microns.</p

Slow fusion and rapid movement of mitochondria within the same neurite.

<p>Time lapse images were taken every 10 seconds for 30 minutes at 0 h, 5 h, and 21 h following the marking of a subpopulation of mitochondria within a living neurite (56 div). The data are presented as kymographs, and show a progressive fusion of all of the mitochondria under observation with organelles originating from the proximal (cell body) and distal sides of the process. At each time point a limited number of mitochondria move during the time lapse imaging. After 21 hours, moving organelles appear to be the product of fusion events.</p

Characterization of motor neurons in spinal cord cultures.

<p>Antibodies were used to confirm the identity of the large neurons growing in the spinal cord cultures (24–29 div). A) A motor neuron (arrow) identified by staining for the non-phosphorylated neurofilament epitope recognized by SMI-32 mAb (green), and a polyclonal antibody against MAP2 (red). The presumptive axon is marked by an asterisk. B) A motor neuron (arrow) visualized with an anti-beta tubulin III antibody (green) does not express the phosphorylated neurofilament epitope recognized by SMI-312 mAb (red), a marker used to identify axons. Nuclei are labeled with DAPI. Scale bars correspond to 50 microns and 20 microns (higher power insets) respectively. C) An infected motor neuron immunostained with SMI-32 mAb (red) showing mitochondria expressing mitoKaede fluorescent protein (green). Arrows point to a close association of mitochondria with neurofilaments. Scale bar corresponds to 20 microns. D) A representative recording of spontaneous activity from an infected spinal cord motor neuron.</p

Bursts as a unit of neural information: selective

communication via resonanc