Using fly genetics to dissect the cytoskeletal machinery of neurons during axonal growth and maintenance

Abstract

SummaryThe extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. For this, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream of these signals is implemented by their actin and microtubule cytoskeleton. In the first part of this Commentary, we discuss the fundamental roles of the cytoskeleton during axon growth. We present the various classes of actin-and microtubule-binding proteins that regulate the cytoskeleton, and highlight the important gaps in our understanding of how these proteins functionally integrate into the complex machinery that implements growth cone behaviour. Deciphering such machinery requires multidisciplinary approaches, including genetics and the use of simple model organisms. In the second part of this Commentary, we discuss how the application of combinatorial genetics in the versatile genetic model organism Drosophila melanogaster has started to contribute to the understanding of actin and microtubule regulation during axon growth. Using the example of dystonin-linked neuron degeneration, we explain how knowledge acquired by studying axonal growth in flies can also deliver new understanding in other aspects of neuron biology, such as axon maintenance in higher animals and humans.

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…Neurons cultured in this way reproduce an impressive range of neuronal in vivo-like properties, including characteristic cytoskeletal organizations and dynamics or physiologically active presynaptic structures, both closely resembling descriptions from vertebrate neurons (Küppers, Sánchez-Soriano, Letzkus, Technau, & Prokop, 2003;Küppers-Munther et al, 2004;Sánchez-Soriano et al, 2010). Since the same combinatorial genetic strategies to combine mutations and/or transgenic elements can be applied in primary neurons just like in vivo (Beaven et al, 2015;Prokop et al, 2013), cultures offer important additional experimental opportunities: First, some of the readouts available in primary neurons are far more detailed than can be achieved in vivo, increasing the resolution of mechanistic studies. Second, genetic manipulations especially of the cytoskeleton often cause in vivo phenotypes so severe that they cannot be interpreted, whereas neurons cultures from such embryos still allow sensible experimentation.…”

“…One important strength of the fly model is the ease with which mechanisms and roles of spectraplakins can be analyzed in biological contexts in vivo, revealing relevant mechanistic concepts that can then be used for studies in higher organisms (Prokop, Beaven, Qu, & Sánchez-Soriano, 2013). To illustrate these points, we will focus here on a number of in vivo readouts used for functional studies of shot.…”

Mathematical-Computational Simulation of Cytoskeletal Dynamics

“…More recently, growth cones of the fruit fly Drosophila have been established as powerful models accessible to detailed studies of the cytoskeleton and systematic genetic dissections of its various regulators [49,51,55,56]. For example, a systematic study used filopodial number and length as a simple readout to understand the systemic contributions and functional interfaces of seven different actin-binding regulators, which included formins, Ena/VASP, profilin, and capping proteins ( Fig.…”

“…The cytoskeleton comprises mechano-resistant, yet highly dynamic, networks composed of filamentous protein polymers, called actin (thin filaments; diameter of 5-7 nm), intermediate filaments (5-12 nm), and microtubules (thick filaments; 25 nm), which support cell architecture and dynamics [20]. There is virtually no cell function that does not depend on the cytoskeleton, yet the number of essential proteins binding and regulating the cytoskeleton is surprisingly low [15,49]. This is possible because the same cytoskeletal regulating proteins can be employed in different contexts, contributing to very distinct cytoskeletal networks and dynamics.…”

“…This is possible because the same cytoskeletal regulating proteins can be employed in different contexts, contributing to very distinct cytoskeletal networks and dynamics. Therefore, genetic defects of any of these components will have multiple (pleiotropic) effects, and the many human diseases caused by such defects [49] are by nature complex systemic phenomena. Furthermore, cytoskeletal dynamics are the cause and consequence of both chemical interactions and physical forces, and its analysis requires thinking at the interface of biochemistry and biomechanics [7].…”