Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development

Abstract

Axonal growth and targeting are fundamental to the organization of the nervous system, and require active engagement of the cytoskeleton. Polymerization and stabilization of axonal microtubules is central to axonal growth and maturation of neuronal connectivity. Studies have suggested that members of the tubulin polymerization promoting protein (TPPP, also known as P25α) family are involved in cellular process extension. However, no in vivo knockout data exists regarding its role in axonal growth during development. Here, we report the characterization of Ringmaker (Ringer; CG45057), the only Drosophila homolog of long p25α proteins. Immunohistochemical analyses indicate that Ringer expression is dynamically regulated in the embryonic central nervous system (CNS). ringer-null mutants show cell misplacement, and errors in axonal extension and targeting. Ultrastructural examination of ringer mutants revealed defective microtubule morphology and organization. Primary neuronal cultures of ringer mutants exhibit defective axonal extension, and Ringer expression in cells induced microtubule stabilization and bundling into rings. In vitro assays showed that Ringer directly affects tubulin, and promotes microtubule bundling and polymerization. Together, our studies uncover an essential function of Ringer in axonal extension and targeting through proper microtubule organization.

Mutations in ACTL6B Cause Neurodevelopmental Deficits and Epilepsy and Lead to Loss of Dendrites in Human Neurons

“…We were interested in those genes that showed significant changes between D0 and D5 in the ChIP-seq data and which also showed significant change in the RNA-seq data between D0 and D5 (within-subjects); also, we selected those genes that showed RNA-seq differences between mutant cells and control cells at D5 (Table S2). We highlight TPPP (Table S4), a microtubule-binding protein involved in cell process extensions, 27,28 and FSCN1, which has been shown to be involved in neurite shape and trajectory in prior studies in mice. 29 Due to their biological function and significance levels in our experiments, we chose to use TPPP and FCSN1 expression levels to assess the external validity of our findings.…”

Tubulin Polymerization Promoting Protein, Ringmaker, and MAP1B Homolog Futsch Coordinate Microtubule Organization and Synaptic Growth

“…Apart from the classical MAPs, there is also a unique family of highly conserved but not well-understood tubulin-binding proteins called the T ubulin P olymerization P romoting P roteins (TPPPs) that are implicated in the modulation and coordination of dynamics and stability of the MT network (Hlavanda et al, 2002; Tirián et al, 2003; Oláh et al, 2013) and display extensive MT bundling and polymerization abilities (Hlavanda et al, 2002; Lehotzky et al, 2010; Tokési et al, 2010; Zotter et al, 2011; Mino et al, 2016; Szabó et al, 2017). In addition to MT bundling activity, TPPPs have been implicated in regulating the levels of acetylation of MT network (Tokési et al, 2010; Szabó et al, 2017).…”

“…Although biochemical and in vitro studies linking TPPP to MT network are known, no mammalian in vivo knockout studies have been reported so far to address its role in nervous system development and function. Recently, from a large scale forward genetic screen, we discovered the Drosophila homolog of human TPPP named Ringmaker (Ringer; Mino et al, 2016). Ringer is a major regulator of axonal MT organization and functions in axon extension (Mino et al, 2016).…”

“…Recently, from a large scale forward genetic screen, we discovered the Drosophila homolog of human TPPP named Ringmaker (Ringer; Mino et al, 2016). Ringer is a major regulator of axonal MT organization and functions in axon extension (Mino et al, 2016). Similar to Ringer, TPPP in zebrafish has been implicated in axon outgrowth (Aoki et al, 2014; Orosz, 2015).…”

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

“…TPPP is crucial for microtubule organisation in the brain and for local microtubule nucleation and myelin sheath elongation 65 . In Drosophila, TPPP mutants (also known as Ringmaker) exhibit defects in axonal extension 66 . In mammals, it is primarily expressed in oligodendrocytes where it stabilises microtubule networks, and its depletion in progenitors inhibits oligodendrocyte differentiation 61 .…”