Functional characterisation of filamentous actin probe expression in neuronal cells

<div><p>Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.</p></div

Summary of quantitative morphological analysis.

<p>Summary of quantitative morphological analysis.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [EGFP control and Lifeact-GFP(1)].

<p>Representative images of neurons transfected with EGFP (A-D) or Lifeact-GFP(1) (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [Lifeact-GFP(2) and Lifeact-EGFP].

<p>Representative images of neurons transfected with Lifeact-GFP(2) (A-D) or Lifeact-EGFP (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Quantitative analysis of axonal morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Neurons transfected with Lifeact-GFP(1) show decreased total axon length (A), decreased primary axon total length (B) and decreased total length of primary axon branches (D), compared to EGFP control. Neurons transfected with Lifeact-EGFP and Utr261-EGFP show also show decreased primary axon total length (B). Neurons transfected with Lifeact-GFP(2), Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in axonal morphology when compared to EGFP control (A-F). Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Quantitative analysis of dendritic morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Compared to EGFP control, neurons transfected with Lifeact-GFP(2) showed significant decreases in the total length of dendritic trees (B), the mean length of primary dendritic shaft (C), the mean length of primary dendritic branches (E) and the mean length of dendritic trees (H). Neurons transfected with Utr261-EGFP showed an increase in the number of primary dendritic branches (D) and mean number of primary dendritic branches per dendritic tree per cell (G), compared to EGFP control. Neurons transfected with Lifeact-GFP(1), Lifeact-EGFP, Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in dendritic morphology when compared to EGFP control (A-H) Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Sholl analysis of dendritic complexity of mouse hippocampal neurons transfected with filamentous actin tracking probes.

<p><b>Dendritic complexity was</b> measured as number of intersections per shell as a function of distance from the soma. A Neurons transfected with Lifeact-GFP(1) displayno significant changes in dendritic compliexity compared to EGFP control. Lifeact-GFP(2) transfected neuron consistently had the lowest number of intersections per shell from 0–60 μm from the soma. Lifeact-EGFP transfected neurons showed an increase in dendritic complexity from 0–20 μm and a decrease from 20–60 μm compared to EGFP control. Lifeact-7-mEGFP transfected neurons showed an increase in dendritic complexity from 0–30 μm and a significant decrease from 30–40 μm. Utr261-EGFP also showed an increase in dendritic complexity from 0–30 μm. F-tractin-EGFP transfection resulted in increased dendritic complexity at 0–20 μm from the cell soma. Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by two-way ANOVA with Tukey's test for multiple corrections. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significant difference between EGFP and each actin tracking probe is indicated by the following symbols: Lifeact-GFP(2) = #, Lifeact-EGFP = §, Lifeact-7-mEGFP = £, Utr-261-EGFP = ⁺, F-tractin-EGFP = ‡.</p

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<p>Translation of mRNA into protein is an evolutionarily conserved, fundamental process of life. A prerequisite for translation is the accurate charging of tRNAs with their cognate amino acids, a reaction catalyzed by specific aminoacyl-tRNA synthetases. One of these enzymes is the aspartyl-tRNA synthetase DARS, which pairs aspartate with its corresponding tRNA. Missense mutations of the gene encoding DARS result in the leukodystrophy hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL) with a distinct pattern of hypomyelination, motor abnormalities, and cognitive impairment. A thorough understanding of the DARS expression domains in the central nervous system is essential for the development of targeted therapies to treat HBSL. Here, we analyzed endogenous DARS expression on the mRNA and protein level in different brain regions and cell types of human post mortem brain tissue as well as in human stem cell derived neurons, oligodendrocytes, and astrocytes. DARS expression is significantly enriched in the cerebellum, a region affected in HBSL patients and important for motor control. Although obligatorily expressed in all cells, DARS shows a distinct expression pattern with enrichment in neurons but only low abundance in oligodendrocytes, astrocytes, and microglia. Our results reveal little homogeneity across the different cell types, largely matching previously published data in the murine brain. This human gene expression study will significantly contribute to the understanding of DARS gene function and HBSL pathology and will be instrumental for future development of animal models and targeted therapies. In particular, we anticipate high benefit from a gene replacement approach in neurons of HBSL mouse models, given the abundant endogenous DARS expression in this lineage cell.</p

Peptide Nanofiber Substrates for Long-Term Culturing of Primary Neurons

The culturing of primary neurons represents a central pillar of neuroscience research. Primary neurons are derived directly from brain tissue and recapitulate key aspects of neuronal development in an in vitro setting. Unlike neural stem cells, primary neurons do not divide; thus, initial attachment of cells to a suitable substrate is critical. Commonly used polylysine substrates can suffer from batch variability owing to their polymeric nature. Herein, we report the use of chemically well-defined, self-assembling tetrapeptides as substrates for primary neuronal culture. These water-soluble peptides assemble into fibers which facilitate adhesion and development of primary neurons, their long-term survival (>40 days), synaptic maturation, and electrical activity. Furthermore, these substrates are permissive toward neuronal transfection and transduction which, coupled with their uniformity and reproducible nature, make them suitable for a wide variety of applications in neuroscience

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

<div><p>The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.</p></div