application-dna-specific-stain-methyl-green-fluorescent-labeling

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Institut Pasteur de Montevideo

Universidad de la República

MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development.

Identification of the chicken MARCKS phosphorylation site specific for differentiating neurons as Ser 25 using a monoclonal antibody and mass spectrometry.

Myristoylated alanine-rich kinase C substrate (MARCKS), an actin-binding protein, is involved in several signal transduction pathways. It is susceptible to be phosphorylated by protein kinases as protein kinase C and some proline-directed kinases. These phosphorylations differently modulate its functions. We previously showed that a phosphorylation at its Ser25 (S25p-MARCKS) in chickens is a signature of this ubiquitous protein in neuron differentiation. To gain insight into the possible involvement of MARCKS in late retinal histogenesis, we compared the developmental expression patterns of the total protein and its S25p variants. Here we show that the most outstanding modifications occur at the outer retina, where S25p disappears at the end of embryonic development and where MARCKS is missing in adults. These results suggest diverse functional specializations in the different retinal layers.

MARCKS in advanced stages of neural retina histogenesis.

The availability of new vital markers and the improvements in time-lapse video microscopy have increased our ability to observe developmental events in the retina while they are happening. Using this approach, advances have been made in the understanding of important issues such as how division patterns relate to cell fate determination, how post-mitotic progenitors reach their correct retinal layers, and finally, how retinal ganglion cell axons navigate out of the retina and to their final targets in the midbrain.

Time-lapse analysis of retinal differentiation.

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.

Polarization and orientation of retinal ganglion cells in vivo.

A little over the last 10 years, zebrafish research in Latin America has been silently maturing, with a community that has grown both through local initiatives as well as because of returning expatriates who were trained in U.S. and European zebrafish laboratories. Now numbering over 20 groups in four countries, principal investigators from these laboratories met in December 2010 to inaugurate the Latin American Zebrafish Network (LAZEN). The meeting got underway with scientific presentations by the lead investigators of each represented laboratory, as a way to gauge the breadth of zebrafish research in the region. Developmental biology of sensory systems, organogenesis, regeneration, and metabolism were well-represented topics, and there were also groups initiating work on applied uses of the zebrafish model (molecule screens, environmental monitoring, and disease models). However, the principal aim of the gathering was to diagnose the difficulties faced by investigators in the different countries and to search for common strategies to resolve them. As an outcome of the encounter, a coordinating committee was elected and future activities were planned, including periodic meetings similar to those held in other regions of the world.

First meeting of the latin american zebrafish network.

How the site of axon emergence is specified during neural development is not understood. Previous studies disagree on the relative importance of intrinsic and extrinsic mechanisms. The axons of retinal ganglion cells (RGCs) emerge basally in vivo, yet because RGCs develop from polarized neuroepithelial cells within a polarized environment, disentangling intrinsic and extrinsic influences is a challenge. We use time-lapse imaging to demonstrate that Laminin acting directly on RGCs is necessary and sufficient to orient axon emergence in vivo. Laminin contact with the basal processes of newborn RGCs prevents the cells from entering a stochastic Stage 2 phase, directs the rapid accumulation of the early axonal marker Kif5c560-YFP, and leads to the formation of axonal growth cones. These results suggest that contact-mediated cues may be critical for the site of axon emergence and account for the differences in cellular behavior observed in vitro and in vivo.

The oriented emergence of axons from retinal ganglion cells is directed by laminin contact in vivo.

MARCKS is a ubiquitous actin-binding protein, with special functions in the development of the central nervous system. We have previously described a neuronal-specific isoform, phosphorylated at serine 25 (S25p-MARCKS), which is present very early during neuronal differentiation in the chick retina. However, very little is known about MARCKS expression or functions in the peripheral nervous system (PNS). In the present work, we analyzed migrating PNS precursor cells in the chick embryo, particularly those originating from the neural crest, and found that they all express a high amount of MARCKS and that a subpopulation of them also contained S25p-MARCKS from early developmental stages. MARCKS protein was also found in dorsal root and trigeminal ganglia during embryo development. Not only is the protein present in these structures but it is also phosphorylated in differentiating neurons with a maximal signal on the ganglion periphery, where neurogenesis is occurring. In conclusion, MARCKS is present and phosphorylated at early stages during the differentiation of PNS cells and precursors, indicating that it might also be important for the differentiation of these tissues.

Early phosphorylation of MARCKS at Ser25 in migrating precursor cells and differentiating peripheral neurons.

MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.

A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.

Structural characterization of a neuroblast-specific phosphorylated region of MARCKS.

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.

Flavio R. Zolessi

Institut Pasteur de Montevideo

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos