Use fly genetics for study axonal grownth

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JournalofCellScience

Using fly genetics to dissect the cytoskeletalmachinery of neurons during axonal growth andmaintenance

Andreas Prokop*, Robin Beaven, Yue Qu and Natalia Sanchez-Soriano*

Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

*Authors for correspondence ([email protected];[email protected])

Journal of Cell Science 126, 23312341 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126912

Summary

The extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. Forthis, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream ofthese signals is implemented by their actin and microtubule cytoskeleton. In the first part of this Commentary, we discuss thefundamental 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 thecomplex 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 ofcombinatorial genetics in the versatile genetic model organism Drosophila melanogasterhas started to contribute to the understandingof actin and microtubule regulation during axon growth. Using the example of dystonin-linked neuron degeneration, we explain howknowledge acquired by studying axonal growth in flies can also deliver new understanding in other aspects of neuron biology, such asaxon maintenance in higher animals and humans.

Key words:Growth cone, Cytoskeleton, Axon, Drosophila, Actin, Microtubule, Brain disorders

Introduction

The cytoskeleton consists of actin, intermediate filaments (IFs) and

microtubules (MTs), and has fundamental roles in virtually everyfunction of cells, including cell division, motility, adhesion, signalling,

endocytic trafficking and transport, as well as in the regulation of celland organelle shapes and their distributions (Akhmanova and Stearns,

2013). Naturally, this also applies to the nervous system where the

cytoskeleton has essential roles during the development, function,

regeneration and degeneration of neurons. Of the more than 200 entries

on the OMIM (Online Mendelian Inheritance in Man) database forgenes encoding cytoskeleton-associated proteins, 44% have indexed

links to human disorders, and 54% of these are linked to nervous

system disorders, including neurodevelopmental disorders (e.g.

lissencephalies, mental retardations), functional disorders (e.g.

deafness) and a wide range of degenerative diseases (seesupplementary material Table S1). Therefore, research of the

cytoskeleton provides unique opportunities to unravel fundamentalmechanisms of the nervous system, both in health and disease.

It is currently little understood how cytoskeleton-associated

proteins contribute to cellular mechanisms of neurons. For

example, the systemic importance of certain MT-binding

proteins, such as motor proteins and the structural protein tau,has long been recognised from their involvement in a broad

spectrum of neurodegenerative diseases, and we know their

principal molecular functions. However, we have yet to explain

conclusively how they function within normal or diseased

neurons (Hirokawa et al., 2010; Morris et al., 2011). Here, we

argue that this is best performed starting with processes for whichthere is already an extensive body of knowledge about the

cytoskeleton, in particular the growth of axons.

Axons are slender neuronal extensions, which can be several

metres long, and are often arranged into nerves or nerve tracts

that provide essential information highways in the nervous

system. The proper function of nervous systems requires axons to

grow and wire up correctly during development or regeneration,

and these connections need to be maintained for decades in the

ageing body. Here, we review the current concepts for the

cellular roles that actin and MTs have in axons and discuss

important gaps in our understanding of how the cytoskeleton is

regulated to this end. We argue that genetics provides important

means to advance this understanding, and explain how studies

using the genetic model organism Drosophila melanogastercanmake important contributions. We review recent advances with

regard to actin and MT regulation during axonal growth in the fly

and provide an example of how mechanistic understanding in one

context can be used to identify mechanisms underpinning other

processes, such as axon maintenance and neurodegeneration.

The organisation of the cytoskeleton in axons

Mature axons are the longest cellular entities that are produced by

animals. They propagate electrical messages, which travel from

the neuronal cell bodies towards the synaptic connections with

their distant target cells, often several metres away. To maintain

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this unusual architecture, shafts of axons contain prominentbundles of parallel MTs, which provide structural support as well

as the tracks for the vital long-distance cargo transport(Twelvetrees et al., 2012). Axonal MTs are discontinuous and

are usually not attached to the centrosome (the main microtubuleorganizing centre of most cells); their plus ends mostly, but not

entirely, point distally (Fig. 1A) (Ahmad et al., 1999; Baas and

Lin, 2011;Basto et al., 2006;Stiess et al., 2010). The cortex ofaxon shafts is lined by short stabilised actin filaments ( Fig. 1A),which are organised into repetitive rings by the scaffolding

protein spectrin and linked to the axonal cell membrane through

ankyrins (Xu et al., 2013). Axons also contain a number ofdifferent IF proteins that can regulate the specific diameters of

different axon classes (Perrot and Eyer, 2009).

Axons are usually maintained for the lifetime of an organism,

i.e. for decades in humans. It is therefore not surprising that axonaldecay is a characteristic feature in the ageing brain (Marner et al.,

2003). Sites of severe disorganisation of axonal MT bundles

(referred to as diverticula) are considered an important trigger ofsuch axon loss (Adalbert et al., 2009;Fiala et al., 2007). Axonal

MT bundles are believed to be stabilised through structural MT-binding proteins (MTBPs), such as tau or MAP1B (Chilton and

Gordon-Weeks, 2007). At the same time, there seems to be asteady and dynamic MT turnover, as suggested by continued MT

polymerisation events in mature axons (Kollins et al., 2009). Suchdynamics might allow for structural plasticity, such as shortening

or extension of the axon shaft (Kuppers-Munther et al., 2004;Lamoureux et al., 2010; Rajagopalan et al., 2010), pruning andregrowth of axons or formation of new side branches (Letourneau,

2009), which are required for rewiring processes during learningand memory formation or the regeneration of damaged axons

(Bradke et al., 2012;Saxena and Caroni, 2007).

Cellular roles of the cytoskeleton during axonal

development

During development, axons are initiated as small neuriticprotrusions on the surface of neuronal cell bodies. For this,

MTs in the neuronal cell bodies become stabilised and pushbeyond the cell cortex (Brandt, 1998). This is assisted by

dynamic rearrangements of the cortical actin cytoskeleton toremove barrier function (Flynn et al., 2012; Neukirchen and

Bradke, 2011; Saengsawang et al., 2012) and the formation offilopodia, which provide membrane protrusions into which MTscan extend (Dent et al., 2007;Goncalves-Pimentel et al., 2011).

In typical vertebrate neurons, several neurites are formedsimultaneously on the cell body, but only one of these is

selected as the axon. This selection process (referred to as neuronpolarisation) involves signalling cascades leading to the targeted

Filopodia

Engorging

filopodium

Engorging filopodium

Cortical actin

Cortical actin rings

F-actinlattice

F-actinbundle

F-actinarc

LamellipodiumSoma

Axon

CentrosomeMT polymerisation

F-actin backflow

Repulsivecue

MT-destabi-lising drug

MT-stabilisingdrugElectric field

MTF-actinlattice

F-actinbundle

StabilisedMT

Attractivecue

or

or

or or

Growthcone

Nucleus

A

B

E F-act F F-actC Splayed MT D F-act

New axon segment

Key

Fig. 1. Principal organisation androles of actin and

MTs in growing axons. (A) The figure shows the

principal distribution and organisation of MTs and F-

actin networks in the soma, axon and growth cone of a

neuron that is exposed to an attractive guidance cue.

Thetip of the axonalMT bundle forms the central zone

of the growth cone from where splayed MTs emanate

into the actin-rich peripheral zone (Dent et al., 2011).

(B) Directed axon extension (growth cone turning).

Splayed MTs become preferentially stabilised in acertain direction through guidance cues [or

experimentally through unilateral exposure to

MT-(de-)stabilising drugs or the application of an

electrical field]. Additional MTs follow in that

direction, leading to engorgement of membrane

structures in thatarea and, eventually, the establishment

of a new axon segment (Buck and Zheng, 2002;

Lowery and Van Vactor, 2009;McCaig et al., 2002;

Sabry et al., 1991;Tanaka and Kirschner, 1995;

Tanaka and Kirschner, 1991). (C) Growthcone turning

requires splayed MTs: low doses of MT-stabilising

drugs suppress the occurrenceof splayed, side-stepping

MTs(splayedMT2). Growthcones that are treated this

way fail to show normal turning behaviour at repulsive

borders and stall instead (Challacombe et al., 1997;

Williamson et al., 1996). (D,E) Growth cone turning

requires F-actin. When F-actin is destabilised through

drugs (F-act2), MTs tend to be bundled and growth

cones fail to turn at repulsive borders (Challacombe et

al., 1996); they also no longer respond to unidirectional

application of MT-stabilising drugs(Buck and Zheng,

2002). (F) Growth cone turning towards the cathode in

electrical fields (galvanotropism) does not require F-

actin (McCaig, 1989); this turning could be mediated

by MTBPs, which are attracted to the cathode through

positive net-charge of their MT-binding domains, thus

dragging the MTs along with them.

Journal of Cell Science 126 (11)2332


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localisation of cell polarity factors (Barnes and Polleux, 2009)and also depends on the specific stabilisation of MTs in theselected neurite (Brandt, 1998;Chen et al., 2013).

Once established, axons grow along stereotypic paths to theirtarget cells, a process that is implemented by prominent hand-shaped growth cones at the axon tips. Growth cones are guidedtowards their specific target areas and cells through precise

spatio-temporal patterns of chemical signals that are arrangedalong their paths (Tessier-Lavigne and Goodman, 1996). Manyguidance cues and their receptors have been identified and shownto mediate repulsion or attraction (Araujo and Tear, 2003;Huberet al., 2003), thus controlling growth cone behaviours, such asgrowth velocity, pausing, turning, retraction or collapse. Thesemorphogenetic movements downstream of guidance signallingare implemented by the prominent actin and MT cytoskeleton ofgrowth cones (Fan et al., 1993), which is arranged in a particularspatial geometry.

The geometry of growth cones can be subdivided into an actin-rich peripheral zone and a MT-rich central zone (Fig. 1A), andthe area in which MTs and F-actin prominently overlap is oftenreferred to as the transition zone. The central zone of growth

cones comprises the tips of the axonal MT bundles, and from heresingle MTs splay out into the peripheral zone. The peripheralzone consists of actin-rich membrane protrusions, which includefinger-like filopodia (containing parallel F-actin bundles) andveil-like lamellipodia (containing F-actin lattices; Fig. 1A).Filopodia and lamellipodia sense guidance cues by presentingtheir receptors on their surfaces. They undergo constant shapechanges, driven by continuous assembly of actin filaments at theleading edges, the retrograde flow of these filaments, and theirdepolymerisation and recycling further back in the transitionzone (Box 1). This actin dynamics allow filopodia to reach outinto the growth cone environment and act as signalling andadhesion sensors (Chien et al., 1993; Davenport et al., 1993;Dwivedy et al., 2007; Mattila and Lappalainen, 2008). They

allow lamellipodia to act as dynamic sites of substrate adhesionand myosin-II-driven contraction and force generation, which areimportant in influencing MT behaviours during guided axonextension (Box 2).

In current models of growth cone guidance (Fig. 1B) (Dentet al., 2011; Lowery and Van Vactor, 2009), external signalstrigger the directional stabilisation of single MTs in the

peripheral zone. These stabilised MTs are then joined by theextension of the bulk of MTs from the central domain (a processreferred to as engorgement), thus giving rise to a new axonsegment. This elongation step is completed when lamellipodiaand filopodia relocate distally to the newly formed axon tip(Fig. 1B). In this model, MTs are the essential implementers ofaxon extension, whereas F-actin is required for the directionalityof this extension (through influencing MT behaviours; Box 2).Accordingly, blocking MT dynamics inhibits axon growth(Letourneau and Ressler, 1984; Sanchez-Soriano et al., 2010;Tanaka et al., 1995), whereas the pharmacological or geneticinhibition of actin polymerisation, either in cultured neurons orinvivo, does not usually suppress axonal growth per se, butabrogates growth cone turning and pathfinding (Fig. 1D,E) (Buckand Zheng, 2002; Challacombe et al., 1996; Chien et al., 1993;Kaufmann et al., 1998; Marsh and Letourneau, 1984; Sanchez-Soriano et al., 2010). This said, a directional movement of growthcones does not always require F-actin (Fig. 1F), as somesignalling mechanisms can target MTs directly. For example,

GSK3-mediated phosphorylation of the MTBPs APC1, MAP1B

and/or CLASP is known to directly regulate MT stability in

growth cones (Hur et al., 2011; Lucas et al., 1998; Purro et al.,

2008).

Box 1. ABPs and MTBPs as essential regulators ofcytoskeletal dynamics

MTs are stiff hollow tubes typically formed by 13 protofilaments

(filamentous polymers ofa- andb-tubulin heterodimers) that grow

and shrink primarily at their plus ends. Actin filaments are more

flexible double-helical chains of actin monomers that maintain their

equilibrium mainly by growth at plus ends and shrinkage at minus

ends (Fletcher and Mullins, 2010; Hawkins et al., 2010; Stricker

et al., 2010). Both have in common that they are polymers of

repetitive building blocks (black helmet-like symbols in the figure)

that are assembled in a polar fashion. Their dynamics are

regulated in comparable ways through the following different

classes of actin-binding proteins (ABPs) and MT-binding proteins

(MTBPs) as illustrated in the box figure.

(1) New actin filaments or MTs are generated through nucleation

factors that catalyse the transition from mono- or oligomers to

polymers; (2) plus-end dynamics, such as (de-)polymerisation,

stabilisation, directionality and targeting is regulated by plus-end-

binding proteins; (3) nucleation or (de-)polymerisation processes

are further regulated by proteins that bind actin or tubulin

monomers or oligomers and determine their availability, for

example, SCAR or N-WASP activates Arp2/3 nucleation, APC1cooperates with mDia1 in nucleation, stathmin sequesters tubulin,

profilin enhances actin polymerisation by ENAH (Bear and Gertler,

2009; Okada et al., 2010; Pollitt and Insall, 2009; Steinmetz,

2007); (4) proteins that bind along actin filaments or MTs can

stabilise them against depolymerisation, cross-link them into

bundles and/or networks, and/or link them to other cytoskeletal

components, organelles or the cortex; (5) minus-end-binding

proteins regulate stability versus (de-)polymerisation; (6,7) plus-

and minus-end-directed motor proteins mediate filament

contraction, the sliding along other cellular structures or

filaments, or the transport of cargo (C in the figure) along F-actin

or MTs; (8) different classes of proteins can sever or actively

depolymerise F-actin or MTs, for example, cofilin depolymerises or

severs pointed ends of F-actin, type 13 kinesins depolymerise MTs

from the plus end, and katanin or spastin sever MT shafts ( Paket al., 2008; Conde and Caceres, 2009); (9) post-translational

modifications (PTM) influence F-actin or MT stability and the

interaction with certain ABPs and MTBPs (Fukushima et al., 2009;

Janke and Bulinski, 2011;Terman and Kashina, 2013).

91

2

6

7

85

4

3

33

C

CPTM

Nucleation

G-actin or-tubulin-tubulin

Polymer/network regulation

Key

Cytoskeletal machinery of neurons 2333


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The importance and challenge of understanding

cytoskeletal machinery

Clearly, the cytoskeleton has essential roles during axonal

growth, but we still do not understand how it is regulated to

perform these functions. Different classes of actin-binding

proteins (ABPs) and MTBPs are known to regulate cytoskeletaldynamics (Box 2); many of these are expressed in neurons

(Table 1), and we often have a reasonable understanding of their

molecular mechanisms, at least in vitro. However, we do not

understand how these individual regulators functionally integrate

into the common cytoskeletal machinery that ultimately

implements neuronal behaviours. This deficit also leaves us

with little means to explain the cellular aberrations causedby mutations of genes encoding ABPs or MTBPs that have

been linked to brain disorders, including disorders of

neurodevelopment and axonal growth (supplementary material

Table S1) (Nugent et al., 2012;Tischfield et al., 2011).

The need to understand cytoskeletal regulation at the cellularlevel in neurons and non-neuronal cells alike is a current problem(Fletcher and Mullins, 2010;Insall and Machesky, 2009). This isclearly reflected by the number of review articles that attempt to

propose models explaining the functional integration ofcytoskeletal regulators (e.g. Conde and Caceres, 2009; Dentet al., 2011; Gallo, 2013; Lowery and Van Vactor, 2009; Pak

et al., 2008). For example, there are mechanistic models aimingto explain how different classes of ABPs functionally combineduring filopodia formation (Mattila and Lappalainen, 2008) andmodels based on end-binding (EB) proteins that propose how anincreasing number of MT-plus-end-associating proteins canregulate MT dynamics (Akhmanova and Steinmetz, 2008;Akhmanova and Steinmetz, 2010) (see also Fig. 2). Currently,such models depend on knowledge gained from a broad rangeof different neuronal systems, and they therefore remainhypothetical and are difficult to test experimentally.

In this Commentary, we promote strategic considerations that

will help to overcome some of the current shortcomings inexplaining cytoskeletal machinery. We believe that strongeremphasis must be given to studying the various parts of

cytoskeletal machinery consistently in the same neuronsystems, so that their functional links and interfaces can bedirectly assessed. These neuronal systems should not only beamenable to biochemical, biophysical and cell biologicalapproaches, but also to the use of combinatorial genetics,therefore allowing a systematic assessment of different genemanipulations or combinations of mutations in the same cells.Such a strategy will help us to integrate the knowledge we haveabout single cytoskeletal regulators so that we can develophigher-order concepts about the governance of the cytoskeletalmachinery during axon growth or in the context of braindisorders.

Drosophilaas a model to study cytoskeletal machinery

during axonal growthOne organism in which combinatorial genetics has been andcontinues to be most successfully applied to the study of complex

biological problems is the fruit fly Drosophila melanogaster(Keller, 1996;Roote and Prokop, 2013). Work in Drosophila islow cost, impressively fast, and particularly amenable to geneticmanipulations and unbiased genetic screens (Sanchez-Sorianoet al., 2007). Accordingly, Drosophila research has been anincubator for new ideas and concepts in neurobiology, and workin the fly revealed many fundamental molecular and cellularmechanisms that have turned out to be conserved in higheranimals (Bellen et al., 2010). Work in Drosophila has madeimportant contributions to the understanding of axon growth,including the discovery and analysis of proteins involved in

pathfinding, and the discovery of mechanisms underpinningpioneer guidance, nerve branching, target cell recognition andaxonal transport (Araujo and Tear, 2003;Astigarraga et al., 2010;Brochtrup and Hummel, 2011; Landgraf and Thor, 2006;Sanchez-Soriano and Prokop, 2005; Sanchez-Soriano et al.,2007;Zala et al., 2013).

To study the cytoskeletal machinery during axon growth, flyneurons offer a number of useful features. First, most of the

Drosophila ABPs and MTBPs display a high level of sequenceconservation with their mammalian counterparts, and they areusually expressed in the nervous system (Table 1) (Sanchez-Soriano et al., 2007). Second, cytoskeletal regulators tend to

Box 2. How F-actin networks influence MTdynamics in growth cones

N F-actin networks generate membrane protrusions on all sides

of growth cones that can be invaded by MTs in any direction.

This facilitates lateral extension and stabilisation of MTs as an

important prerequisite for growth cone turning (see alsoFig. 1B).

N F-actin networks can influence MT behaviours mechanically

(Lee and Suter, 2008;Schaefer et al., 2002) (see alsoFig. 1A).

Thus, MT extension is antagonised by F-actin backflow or

through the formation of transverse bundles (arcs) that form

road-blocks. MT extension can be promoted and guided

through the formation of radial F-actin bundles as tracks for MT

elongation. MTs can be pushed laterally or medially through

actomyosin contractions. Finally, MT extension can be

channelled into certain directions through coordinated loss of

F-actin from local spots within lamellipodia.

N Actin networks form scaffolds that serve as a platform for ABPs.

Some of these ABPs influence MT behaviours through mediating

actinMT linkage. This linkage can be direct through ABPs that

also contain MT-binding domains [e.g. MAP1B, spectraplakins,

coronin 7 (POD1 in Drosophila), cytoplasmic-linker-associatedproteins (CLASPs) or adenomatous polyposis coli (APC)

(Bouquet et al., 2007;Moseley et al., 2007; Rothenberg et al.,

2003; Sanchez-Soriano et al., 2009; Tsvetkovet al., 2007)]. Other

ABPs are involved in indirect crosstalk with MTs by interacting

with MTBPs. Reported examples are the ABP drebrin that is

linked to the MTBP EB3, the ABP PPP1R9A (better known as

spinophilin) that binds to DCX, IQGAP that interacts with CLIP-

170, or functional interactions between the actin-binding motor

protein myosin II and the MT-associated dynein motor protein

complex (Bielas et al., 2007;Geraldo et al., 2008;Myers et al.,

2006;Swiech et al., 2011).

N Some ABPs can tether actin networks to transmembrane

receptors when they are engaged with their extracellular

ligands. This has been demonstrated for different classes of

transmembrane receptors (Giannone et al., 2009;Moore et al.,2009; Moore et al., 2010). Such linkage of F-actin to the

extracellular environment generates mechanical forces across

the membrane, which can trigger intracellular signalling events

(referred to as the clutch mechanism) that then can influence

actin and MT dynamics (Suter and Forscher, 2000;Suter and

Miller, 2011).



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display minimal redundancy in the fly genome. For example,eliminating the function of Ena/VASP requires a triple knockoutin mouse, whereas only a single gene needs to be removed in

Drosophila(Goncalves-Pimentel et al., 2011;Kwiatkowski et al.,2007). Third, mutant alleles or other genetic tools are often

readily available (McQuilton et al., 2012), and they can usuallybe combined in the same animal in a short time (Goncalves-Pimentel et al., 2011; Koper et al., 2012). Finally,experimentation is fast; axon extension can be studied in flyembryos after only half a day of development, and in primaryneurons after only 6 hours in culture (Prokop et al., 2012;Sanchez-Soriano et al., 2007).

To study cytoskeletal regulation during axonal growth, geneticmanipulations of cytoskeletal regulators can be combined with a

broad spectrum of morphological readouts for axon growth invivo (Sanchez-Soriano et al., 2007), including live imaging ofcytoskeletal dynamics (Mattie et al., 2010;Pawson et al., 2008).This repertoire can be further expanded when makingcomplementary studies in Drosophila primary neurons, whichgive access to quantitative measurements of filopodia andlamellipodia, retrograde F-actin flow, the organisation,stabilisation and polymerisation rates of MTs, as well as axonalgrowth rates (Prokop et al., 2012;Sanchez-Soriano et al., 2010).Such readouts are crucial to unravel how ABPs and MTBPsregulate the cytoskeleton in cells. Notably, cytoskeletal dynamics

in fly neurons are similar to those of other animal neuronsystems, indicating that the underlying regulation is fundamentaland well preserved (Sanchez-Soriano et al., 2010). This said,there will be limitations when using neurons of this little insect

because some aspects are different in vertebrate neurons;for example, the axon length and life time, the absence

of neurofilaments and myelination, and their unipolarorganisation, including the spike-initiating zone, which islocated at the base of dendrites away from the soma and notnear the cell body as is the axon initial segment in vertebrates(Grubb and Burrone, 2010;Sanchez-Soriano et al., 2005). In the

following sections, we will provide examples of work on axonalgrowth in the fly that has contributed new understanding ofcytoskeletal machinery.

Use of the fly to unravel new ABP functions

during axonal growth

Within a few years, work in Drosophila growth cones hasidentified at least four new ABPs as actin regulators in neurons,including Shot, DAAM, Psidin and Canoe. Shot is the only

Drosophila member of the spectraplakin family of actin- andMT-binding proteins with two close homologues in mammals,

MACF1 (also known as ACF7) and dystonin (DST, also knownas BPAG1) (Suozzi et al., 2012). Both Shot and ACF7 promotefilopodia formation (Sanchez-Soriano et al., 2009). In Shot, thisfunction is relevant for axon guidance at the CNS midline, and itrequires binding of the two EF-hand domains to the putativetranslational regulator Extra bases (Exba, also known as eIF5C)(Fig. 2A) (Lee et al., 2007; Sanchez-Soriano et al., 2009), thus

potentially contributing to local translation events in growthcones (Van Horck and Holt, 2008). DAAM (DAAM1 inmammals) is one of four Drosophila formins (Diaphanous,DAAM, Fhos and CG32138) reported to be present in thenervous system (Pawson et al., 2008; Prokop et al., 2011;Sanchez-Soriano et al., 2007). DAAM has now been shown to bethe essential formin in growth cones of embryonic neurons,where it promotes filopodia formation and axonal pathfinding in

Table 1. Examples of evolutionary conserved MTBPs and ABPs in human and fly

Cytoskeleton regulator class MTBPs ABPs

Nucleators c-tubulin ring complex (cTub23Cand other components);doublecortin* (DCX-EMAP)

Arp2/3 complex (Arpc1, Arp3, etc.); formins(DAAM, Fhos, CG32138, dia)

Plus-end-binding proteins MAPFRE/EB* (eb1); MT-plus-end-tracking proteins (+TIPs);doublecortin* (DCX-EMAP); CKAP5/CHTOG/XMAP215*(msps)

ENAH/VASP* (ena); formins (DAAM, Fhos,CG32138, dia); capping proteins* (cpa, cpb);adducins* (hts)

Mono- or oligomer-binding proteins Stathmin/SCG10* (stai); DPYSL2/CRMP2* (CRMP); CKAP5/CHTOG/XMAP215* (msps); doublecortin* (DCX-EMAP);tub-specific chaperone E* (tbce); CLASP* (chb)

WASF/SCAR/WAVE* (SCAR); WASL/N-WASP*(WASp); APC* (Apc); b4-thymosin/TMSB4X*(cib)a; profilin* (chic); twinfilin* (twf)

Shaft stabilisers and cross-linkers MAPT* (tau); MAP2* (tau); MAP1B* (futsch); MACF1*;dystonin* (shot)

Fascin* (sn); a-actinin* (Actn); plastin/fimbrin*(Fim); filamin* (cher); tropomyosin* (Tm1);moesin*, ezrin*, radixin* (Moe); a/b-spectrin* (a/b-Spec); drebrin* (CG10083); coronin* (coro,

pod1); MLLT4/Afadin-6* (cno); CTTN*(Cortactin); PPP1R9A* (spinophilin)b

Minus-end stabilisers c-tubulin ring complex (cTub23Cand other components);CAMSAP1* (Patronin)

Tropomodulin* (tmod)b; Arp2/3 complex (Arpc1,Arp66B, etc.)

Plus-end motors Type 1 kinesins (Khc, Klc, Pat1); type 2 kinesins (Klp64D,Klp68D, Kif3C, Kap3); type 3 kinesins (unc-104, Khc-73)

Myosin XVA* (Myo10A); myosin VA* (didum);myosin II (zip, sqh)

Minus-e nd motors cytopla smic dynein/dynactin (Dhc64C, ctp, sw, Gl, etc.); type13 kinesins/KIF2C/MCAK* (Klp10A, Klp59C, Klp59D)

Myosin VI* (jar)

Severers, de-polymerisers katanin* (Kat60); spastin* (spas); type 13 kinesins/KIF2C/MCAK* (Klp10A, Klp59C, Klp59D)

Cofilin* (tsr); gelsolin* (Gel)

Post-translational modifications(enzymes not listed)c

GTP hydrolysis; acetylation; (poly-) glycylation; (poly-)glutamylation; de-tyrosination; de-glutamylation (D2 tubulin)

ATP hydrolysis; potentially acetylation, arginylation,phosphorylation, etc.

Some proteins fulfil more than one function and are repeated in different positions; terms highlighted with an asterisk are listed in OMIM, other terms use otherdescriptors for protein complexes or classes. Terms in brackets are the orthologous Drosophila genes with known functions and/or prominent expression in thenervous system, as indicated in FlyBase (McQuilton et al., 2012).

aCib represents a triple-repeat of B4-thymosin.bNot declared as orthologues in FlyBase in spite of partial sequence homologies.cAs described byJanke and Bulinski, 2011; Terman and Kashina, 2013.



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the central nervous system (CNS) (Goncalves-Pimentel et al.,2011; Matusek et al., 2008). Psidin (known as NAA25 inmammals) is the auxiliary subunit of the NatB acetylation

complex required for protein acetylation (Stephan et al., 2012). Inaddition, it has been shown that Psidin can act outside this

complex as an ABP that regulates actin dynamics both in neurons

and in non-neuronal cells of Drosophila (Kim et al., 2011;Stephan et al., 2012). These studies show that Psidin antagonises

F-actin stabilisation mediated by Tropomyosin 1 in lamellipodia,which is required for the pathfinding of olfactory neurons in the

fly brain. Canoe and its mammalian homologue MLLT4 (alsoknown as afadin, AFD6) are PDZ-domain-containing ABPs thatlocalise to adherens junctions and have been shown to interact

with profilin (Boettner et al., 2000;Lorger and Moelling, 2006;Sawyer et al., 2009). In Canoe-deficient embryos, axonal

pathfinding at the CNS midline is impaired, and this aberration

correlates with two growth cone phenotypes: an increase infilopodia numbers and a failure to localise Robo proteins

(receptors that are important regulators of midline pathfinding)to filopodia (Slovakova et al., 2012). In this scenario, Canoe

EB1Exba /eIF5C

Tau

DAAM

Arp2/3

Enabled

Profilin

CpA+B

Psisin

Tm1

Sh

ot

Sh

ot

MAP1B

GRDC-tail

MtLSEFSR-rodplakinCH

EB1MTsExba/eIF5CF-actin

Axon extension

FilopodiaLamellipodia

Cortex

Other MTs

Apc

Dynein

Type 13 kinesins

CLIP190

CLASP

XMAP

DCX STMN

Ssp2

Actin binding

MT binding

EB1 binding (+TIP)

Tubulin binding

Other binding

Actin regulatingOther regulatingprocessesSub-machinery

2 Actin dynamics

1 MT plus-end dynamics3 MTshaft

stability

MT growth

-tubulin F-actin

B

A

Key

-tubulin

Interactions

Fig. 2. Roles of Shot in MT regulation at the interface with other classes of ABPs and MTBPs.(A) The functional domains of Shot (below the structure) and

their reported molecular interactions (above the structure). CH, calponin homology domains; plakin, plakin-like domain; SR-rod, spectrin repeat rod; EF, EF-hand

motifs; GRD, Gas2-related domain; MtLS, microtubule tip localisation sequence; C-tail, C-terminal tail. (B) A close-up of a growth cone showing a single MT andthree different aspects of the cytoskeletal machinery (emboxed and numbered), all of which are being studied in fly neurons. (1) Regulation of MT plus-end

dynamics; it includes end-binding proteins (here EB1) which directly bind MT plus ends and recruit MT-plus-end-tracking proteins (+TIPs) (blue arrows; see

Akhmanova and Steinmetz, 2008;Etienne-Manneville, 2010). Shot has to compete with other +TIPs for binding to a limited pool of EB1; when bound to EB1, it

links MT plus ends to F-actin (via its N-terminal CH domains) and guides polymerising MT plus ends in the direction of axon growth. The proper localisation of

Shot at MT plus ends also requires direct association with MTs through its GRD and C-terminal tail. CLASPs and XMAP bind tubulin and promote MT

polymerisation; CLASPs are +TIPs whereas XMAP215 (XMAP) binds MT plus-ends either directly or through +TIPs: Small spindles 2 (Ssp2) in Drosophilaand

Slains in mammals (not shown;Al-Bassam and Chang, 2011;Currie et al., 2011;van der Vaart et al., 2012;Li et al., 2011; Lowery et al., 2010). Stathmins

(STMN) sequester tubulin (Manna et al., 2009), and doublecortin (DCX) binds MT plus ends independently (Bechstedt and Brouhard, 2012). (2) Actin dynamics.

The Shotactin linkage is influenced by actin regulators (red stippled arrows) that coordinate the dynamics and structure of F-actin networks (cortex, lamellipodia,

filopodia; Pak et al., 2008;Xu et al., 2013). In the context of filopodia formation, Shot contributes to the regulation of F-actin through binding of Exba/eIF5C at its

EF-hand motifs (Lee et al., 2007;Sanchez-Soriano et al., 2009). (3) MT shaft stability. In addition, Shot binds along MT shafts and stabilises them independent of

its linkage to F-actin (Sanchez-Soriano et al., 2009;Alves-Silva et al., 2012). Other MT shaft binders are Tau which protects MTs against the MT-severing protein

Katanin in axons (not shown;Qiang et al., 2006), and mammalian MAP1B can cross-link MTs as well as mediate actin-MT linkage during axon growth ( Bouquet

et al., 2007;Riederer, 2007). Tau, MAP1B and Shot (in an EB1-independent mode) influence MT polymerisation kinetics through yet unknown mechanisms

(stippled grey arrow;Alves-Silva et al., 2012;Feinstein and Wilson, 2005;Tymanskyj et al., 2012).



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might regulate the number of filopodia directly through itsfunction as an ABP, or indirectly through acting as a scaffold forRobo receptors, since loss of Robo has been previously shownto lead to an increase in the number of filopodia (Murray andWhitington, 1999).

Use of the fly to study the functional integration

of ABPs during filopodia regulationDrosophila growth cones have been used to carry out loss-of-function analyses of other ABP-encoding genes, including Arpc1(ARP2), Arp3 (ARP3), capping protein A (CAPZA), capping

protein B (CAPZB), chickadee (profilin), enabled (ENAH, alsoknown as VASP), singed (fascin) and tropomyosin 1 (TPM1)(human homologues are given in the brackets) (Goncalves-Pimentel et al., 2011;Kraft et al., 2006;Sanchez-Soriano et al.,2010). All these data and tools can now be combined tounderstand the functional networks of ABPs, as is best illustratedfor studies of filopodia formation. Filopodia are prominent actin-

based structures, and data from numerous studies in a wide rangeof cellular models have led to the formulation of two compositemodels of filopodia formation (Mattila and Lappalainen, 2008).

In the convergent elongation model, Arp2/3 generates actinfilaments in lamellipodia, and ENAH/VASP aggregates their plusends to give rise to elongating filopodial bundles. In the de novonucleation model, formin molecules aggregate to generate

clusters of new actin filaments, which then directly elongateinto parallel filopodial bundles.

Drosophila neurons were used to test whether these two

models co-exist in neurons. Single-mutant analyses of the factorsessential in these models (i.e. Enabled, the formin DAAM anddifferent Arp2/3 complex components) all caused a partial butsignificant reduction in filopodia numbers, in agreement with the

potential co-existence of both models (Goncalves-Pimentel et al.,2011;Matusek et al., 2008). When loss of the Arp2/3 componentSop2 was combined with functional loss of DAAM in the same

neurons (DAAM2/2 Sop22/2 double-mutant neurons), filopodiawere completely absent and F-actin levels were severely depleted(Goncalves-Pimentel et al., 2011). To our knowledge, a geneticcondition that depletes F-actin to this degree has not beenreported for other eukaryotic systems. It indicates that Arp2/3 andDAAM are the prevailing nucleators in embryonic Drosophilaneurons. The fact that the two nucleators enhance each others

phenotypes indicates that they work in distinct complementaryfunctional pathways during filopodia formation. This isconsistent with the biochemical evidence that Arp2/3 andformins nucleate actin filaments through different molecularmechanisms (Chesarone and Goode, 2009; Mattila andLappalainen, 2008). However, it does not yet resolve whetherthese two nucleators contribute to two entirely separate processesof filopodia formation.

To make this distinction, genetic interaction studies usingpartial loss of gene functions are very helpful. For example, inheterozygous gene constellation (i.e. one mutant and one normalgene copy), the levels of the proteins they encode tend to be onlymoderately reduced, and, typically, this reduction is not sufficientto cause a phenotype. Accordingly, neurons that are heterozygousfor DAAM, Sop2 or ena show normal filopodia numbers(Goncalves-Pimentel et al., 2011). However, any combinationsof these heterozygous conditions (transheterozygous mutantneurons: DAAM2/+ Sop22/+ or DAAM2/+ ena2/+ or Sop22/+

ena2/+) become functionally rate-limiting and lead to a strong

reduction in filopodia numbers (Goncalves-Pimentel et al., 2011).These findings are inconsistent with regard to whether twocompletely separate modes of filopodia formation exist, but dosuggest that the pathways downstream of the two nucleatorsconverge at some point. Therefore, these authors suggested amore general model of convergent elongation, in which not onlyArp2/3-derived but also DAAM-derived actin filaments can be

used by Ena to be transformed into filopodial actin bundles(Goncalves-Pimentel et al., 2011). These examples illustrate howsystematic genetic analyses used in Drosophila neurons cancontribute to the formulation of new mechanistic models ofcytoskeletal regulation.

Use of the fly to study mechanisms of actinMT

linkage during axonal growth and maintenance

ActinMT crosstalk is an important aspect of cytoskeletalregulation in growing axons, but its mechanisms are littleunderstood (Box 2). Promising contributions have been made bywork on the above mentioned spectraplakins Shot and ACF7.Besides filopodia-associated phenotypes, we have shown thatloss of Shot in fly neurons and loss of the mouse homologue

ACF7 in mouse neurons result in another conserved phenotype disorganised axonal MT bundles that is coupled with reducedaxon growth (Sanchez-Soriano et al., 2009). Detailed work onShot has shown that these functions depend on actinMT linkage.

Structurefunction analyses have revealed that functions of Shotin MT organisation depend on three simultaneous molecularinteractions (Fig. 2): (1) binding of its N-terminal calponinhomology domains to F-actin, (2) association to MTs throughtwo C-terminal domains (the Gas2-related domain and the

positively charged C-terminal tail), and (iii) binding of C-terminal tail to EB1 (end-binding protein 1) at MT plus ends(Alves-Silva et al., 2012; Bottenberg et al., 2009; Lee andKolodziej, 2002; Sanchez-Soriano et al., 2009). These findingssuggest a model in which Shot binds to the plus ends of

polymerising MTs and guides them along actin structures in thedirection of axon growth (Alves-Silva et al., 2012). This model isfurther supported by the observation that frequent off-trackextensions of MTs are seen during live imaging of shotmutantneurons; this can explain the observed MT disorganisation anddiminished axon growth (off-track MTs are less likely tocontribute to axon extension). The guidance model is alsosupported by shot-like MT-disorganisation phenotypes observedin EB1-deficient, as well as in shot2/+ eb12/+ transheterozygousmutant neurons (Alves-Silva et al., 2012), and, furthermore, isconsistent with proposed roles of ACF7 in guiding MT extensionalong actin stress fibres towards focal adhesions in non-neuronalcells (Kodama et al., 2004).

Notably, the MT guidance model offers a promising molecularplatform for further research. On the one hand, it can be used tounderstand the roles of other cytoskeletal regulators during MTguidance, for example of ABPs or the various proteins that bindand regulate MT plus ends (see details in Fig. 2). On the otherhand, the MT guidance model suggests a potential mechanismcontributing to the maintenance of axons in the mature nervoussystem, as will be explained in the following.

The second mammalian homologue of Shot, dystonin, isstrongly expressed in peripheral neural ganglia (Leung et al.,2001), which, in dystonin mutant mice, undergo a severeneurodegeneration at postnatal stages that is referred to asHSAN (hereditary sensory autonomic neuropathy) (Duchen et al.,



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1963;Duchen et al., 1964). The underlying mechanisms remaininconclusive and were proposed to be associated with roles ofdystonin in intermediate filament organisation and axonaltransport, and in ER or Golgi dysfunction (Ferrier et al., 2013;Young and Kothary, 2007). We propose instead that HSAN

pathology might be primarily caused by defects in MT guidanceand stabilisation on the basis of the following argument. Axonal

MT bundles are believed to be stabilised through structuralMTBPs, such as tau or MAP1B (Fig. 2) (Chilton and Gordon-Weeks, 2007). At the same time, there appears to be a steadyand dynamic MT turnover, as suggested by continued MT

polymerisation events in mature axons (Kollins et al., 2009).Spectraplakins can regulate both functions through twofunctionally conserved domains at their C-terminus: the Gas2-related domain can stabilise MTs against depolymerisation,whereas the C-terminal tail mediates the aforementioned bindingto EB1 required for MT guidance. These domains jointly mediatea robust association along the shaft and at the tip of MTs that isrequired for both MT guidance and stabilisation (Fig. 2) (Alves-Silva et al., 2012; Honnappa et al., 2009; Sun et al., 2001).Absence of either domain in Shot causes severe MT

disorganisation and loss of MT stability in axons of developingfly neurons (Alves-Silva et al., 2012; Sanchez-Soriano et al.,2009) and, in addition, mature sensory neurons of dystoninmutant mice, which are prone to degeneration display severe MTdisorganisation and loss of MT stability (Ferrier et al., 2013;Yang et al., 1999). Consistently, a recently reported frame-shiftmutation in the human dystonin gene functionally deletes theGas2-related domain and the C-terminal tail and causes a severeform of HSAN that mirrors the degenerative pathology of dystoninmutant mice (Edvardson et al., 2012). These data suggest thatspectraplakins perform similar MT-regulating functions during thedevelopment of fly neurons and the maintenance of sensorymammalian neurons. Severe MT disorganisation and instabilitycaused by absence of spectraplakins can, therefore, be expected to

affect vital functions such as axonal transport and impactnegatively on neuronal survival (Sheng and Cai, 2012).

This example shows how functional studies of cytoskeletalregulators during axon growth in the fly can provide new ideasand testable hypotheses for research on other aspects of neuronalcytoskeleton, even in other animals.

Conclusions and future perspectives

In this Commentary, we have discussed how systematic andcombinatorial genetic studies of different ABPs and MTBPs inthe neuronal system of the fly can improve our understandingof cytoskeletal regulation during a biological process such asaxon growth. Considering the high degree of evolutionaryconservation of actin, tubulin and their regulators, theenormous similarities of cytoskeletal dynamics in fly andvertebrate neurons, and the similarities of mutant phenotypesreported so far, we feel that insights gained in the fly system will

be informative for cytoskeletal research in mammalian or othervertebrate neurons.

Understanding the cytoskeletal regulation that underpinscellular behaviours will have important implications andapplications. For example, we still have little understanding ofhow signalling mechanisms instruct the cytoskeleton to influencegrowth cone behaviours (Huber et al., 2003). Knowing how thevarious ABPs and MTBPs that are targeted by particularsignalling events contribute to the cellular cytoskeletal

machinery will greatly help to close this knowledge gap.Understanding these cellular roles of cytoskeletal regulators isalso the first step on the long path to developing mathematicalmodels that can describe the cytoskeletal dynamics that are at the

basis of different cellular processes (Oelz et al., 2008), and it willfacilitate the introduction of systems approaches for cell biologyresearch (Liberali and Pelkmans, 2012). Furthermore, the

principal strategies described here for axon growth can also beapplied to the investigation of the roles and regulation of thecytoskeleton in other relevant areas, such as synapse formation,axonal pruning and Wallerian degeneration, axon regenerationand neurodegeneration. Notably, for all of these processes,suitable Drosophila models have already been established, thushopefully paving the way for rapid progress (Bossing et al., 2012;Fang and Bonini, 2012; Gistelinck et al., 2012; Goellner andAberle, 2012;Jaiswal et al., 2012).

Acknowledgements

We would like to thank Tom Millard, Laura Anne Lowery, PhillipGordon-Weeks, Joszef Mihaly and Erik Dent for their very helpfuland constructive comments on this manuscript.

Funding

Authors on this Commentary were funded by the Biotechnology andBiological Sciences Research Council (BBSRC) [grant numbers BB/D526561/1, BB/I002448/1]. Deposited in PMC for immediate release.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126912/-/DC1

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Use fly genetics for study axonal grownth