© 2020. Published by The Company of Biologists Ltd.

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Cofilin regulates axon growth and branching of Drosophila γ neurons

Sriram Sudarsanam1,2,3, Shiri Yaniv1,3, Hagar Meltzer1,3, and Oren Schuldiner1*

1 Department of Molecular Cell Biology, Weizmann Institute of Sciences, Rehovot,

Israel

2 Current address: The Solomon H. Snyder Department of Neuroscience; Johns

Hopkins University

3 Equal contribution

* Corresponding Author and Lead Contact:

e-mail: oren.schuldiner@weizmann.ac.il

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JCS Advance Online Article. Posted on 9 March 2020

Abstract

The mechanisms that control intrinsic axon growth potential, and thus axon

regeneration following injury, are not well understood. Developmental axon regrowth

of Drosophila mushroom body γ neurons during neuronal remodeling offers a unique

opportunity to study the molecular mechanisms controlling intrinsic growth potential.

Motivated by the recently uncovered developmental expression atlas of γ neurons,

we here focus on the role of the actin severing protein cofilin during axon regrowth.

We show that Twinstar (Tsr), the fly cofilin, is a crucial regulator of both axon growth

and branching during developmental remodeling of γ neurons. tsr mutant axons

demonstrate growth defects both in vivo and in vitro and also exhibit actin rich

filopodial-like structures at failed branch points in vivo. Our data is inconsistent with

Tsr being important for increasing G-actin availability. Furthermore, analysis of

microtubule localization suggests that Tsr is required for microtubule infiltration into

the axon tips and branch points. Taken together, we show that Tsr promotes axon

growth and branching, likely by clearing F-actin to facilitate microtubules protrusion.

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Introduction

The limited regeneration of injured adult neurons within central nervous systems is

due to a combination of inhibitory environments (Tedeschi and Bradke, 2017) and

reduced intrinsic growth ability (Mahar and Cavalli, 2018). Intrinsic growth abilities

decrease with age, concomitant with lower regeneration capacity (Wang et al.,

2007). Therefore, uncovering the factors that determine intrinsic growth potential in

young, developing, neurons, holds the promise of furthering our understanding

regarding factors that normally restrict regeneration.

Neuronal remodeling is a conserved mechanism, which includes the elimination of

axons and synaptic connections, often followed by formation of new connections to

sculpt mature neural networks (Luo and O'Leary, 2005; Yaron and Schuldiner,

2016). Remodeling of the Drosophila mushroom body (MB), a center for olfactory

associative learning, is an excellent model to study developmental axon regrowth

(from now on – regrowth) as neurons need to rapidly change their growth state from

pruning (degeneration) to regrowth (regeneration; Rabinovich et al., 2016). The MB

is comprised of three types of sequentially born neurons - γ, α’/β’ and α/β - of which

only γ-neurons undergo developmental remodeling (Fig 1A; Lee et al., 1999). While

we have previously shown that axon regrowth is a genetically controlled program,

dependent upon the nuclear receptor transcription factors Unfulfilled (UNF; Yaniv et

al., 2012) and Ecdysone-induced protein 75B (E75; Rabinovich et al., 2016), the

molecular machinery that governs growth in this context is largely unknown.

Importantly, we have demonstrated that regrowth is not only molecularly distinct from

initial axon outgrowth, but also shares molecular mechanisms with regeneration

following injury (Yaniv et al., 2012). To continue to dissect the genetic program that

controls axon regrowth, we have recently uncovered the detailed transcriptional

landscape of developing γ-neurons (Alyagor et al., 2018). We found that several

actin regulators exhibit significant expression dynamics during neuronal remodeling,

positioning them as candidates for structural components of axon regrowth.

Axon growth is thought to be driven by growth cones that consist of an actin-rich

peripheral zone with filopodia and lamellipodia, and a microtubule (MT)-rich central

zone. The ability of growth cones to navigate depends on a dynamic interplay

between F-actin and MTs (Blanquie and Bradke, 2018). F-actin dynamics is the

combined result of nucleation, elongation and severing of filaments (Omotade et al.,

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2017; Shekhar et al., 2016). The actin depolymerization factor (ADF)/Cofilin family

consists of small globular proteins (3 in mammals) that depolymerize and/or sever F-

actin filaments, and are also implicated in G-actin sequestration (Bernstein and

Bamburg, 2010; Flynn et al., 2012; Wioland et al., 2017). Counterintuitively,

ADF/Cofilin proteins are known to enhance axon growth and have been implicated in

neurite formation during development (Flynn et al., 2012) as well as growth cone

motility (Endo et al., 2003), branching (Bernstein and Bamburg, 2010; Kanellos and

Frame, 2016) and most recently, axon regeneration (Tedeschi et al., 2019).

Interestingly, actin stabilization by pharmacological manipulation or by mutating

Cofilin slows neurite growth and axon regeneration (Flynn et al., 2012; Tedeschi et

al., 2019). The two main hypotheses as to how cofilin promotes axon extension are:

1) by increasing F-actin assembly, either by ‘actin treadmilling’ (Shekhar and Carlier,

2017) expected to increase the supply of severed G-actin to the growing barbed

ends, or by removal of capping, thus providing new barbed ends available for

elongation, 2) by clearing actin to allow microtubules protrusion (Bradke, 1999).

Twinstar (Tsr, the sole Drosophila cofilin), which was shown to bind actin in vitro and

to promote its depolymerization and severing (Shukla et al., 2018), is highly and

dynamically expressed in MB γ-neurons throughout remodeling (Alyagor et al.,

2018). Here we explore the its role during axon regrowth and branching.

Results and discussion

Twinstar is required for axon growth of MB γ-neurons

We have recently uncovered the expression profiles of developing mushroom body

(MB) γ-neurons at a fine temporal resolution (Alyagor et al., 2018). Out of 126 actin-

related genes in Drosophila, 77 are significantly expressed, 45 in a dynamic pattern

in developing γ-neurons (Fig S1A, data taken from Alyagor et al., 2018), out of which

21 are expressed in a pattern suggestive of a potential role in axon regrowth. In this

study, we decided to focus on the actin filament severing protein twinstar (Tsr, the fly

cofilin) whose expression is upregulated during metamorphosis (Fig S1A) with

maximal values reached at the onset of regrowth (at 24 hours after puparium

formation; APF).

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Tsr has been previously shown to be required for axon growth of both γ and α/β

neurons (Ng and Luo, 2004). Additionally, tsr axons displayed abnormal protrusions

and swellings. However, the nature of these swellings, and whether Tsr is required

for initial axon outgrowth, regrowth, or both, remained unknown. We therefore

generated tsr homozygous mutant clones using the MARCM (mosaic analysis with a

repressible cell marker) technique. In line with previous studies (Ng and Luo, 2004),

MARCM γ-neuron neuroblast clones (NBCs) homozygous for tsrN121 or tsrN96A, both

considered nulls derived from imprecise excision of the same P-element (but not

molecularly defined), display reduced cell numbers and a severe growth phenotype

in which axons stop at the branchpoint (Fig 1E-F, M, S1B). In order to differentiate

between a defect in initial outgrowth or regrowth, we examined clones at 3rd instar

larva (L3) and found that most, but not all, larval tsr γ-axons stall near the peduncular

branch point (Fig 1B-C). The growth defects can be rescued by expressing a full

length Tsr transgene within the mutant cells (Fig D, G, M). To our surprise, NBCs of

the later born α/β neurons appeared normal in tsr mutants (Fig S1C-E), even though

Tsr has previously been implicated in their proper growth (Ng and Luo, 2004). This

inconsistency could be due to the use of different Gal4 drivers (R44E04-Gal4 in this

study vs OK107 in the original paper), and thus labeling different α/β sub-

populations; or due to the timing of the heat-shock induced recombination (L3 here

vs pupae in Ng and Luo).

We next examined single cell clones (SCCs), in which the anatomical resolution is

improved. In contrast to neuroblast clones, tsrN121 SCCs extend their axons normally

at L3 (Fig 1H-I). The fact that SCCs undergo initial growth normally in contrast to

NBCs is likely due to protein and/or RNA perdurance of Tsr, a phenomenon that has

been previously shown (Yu et al., 2013). Briefly, in NBCs, tsr RNA and protein are

diluted by numerous cell divisions, while in SCCs gene deletion occurs during the

last division and thus protein and RNA might still be present.

The fact that tsr SCCs grow normally in larvae (and also at 6h APF, n=17, not

shown) but exhibit a varying degree of growth defects in adult (Fig 1J-K, N, Fig S1F-

J), suggests that Tsr is required for regrowth independently of its requirement during

initial axon outgrowth. Furthermore, overexpressing a WT Tsr transgene (UAS-tsr.N)

within tsrN121 MARCM clones completely rescued the regrowth defect (Fig 1L, N).

However, expression of the phosphomimetic Tsr.S3E, presumed to be inactive (Ng

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and Luo, 2004) did not (Fig 1N, S2B). Interestingly, expression of the non-

phosphorylatable Tsr.S3A, presumed to be constitutively active, resulted in partial

growth rescue (Fig 1N, S2A), suggesting that phosphoregulation of Tsr is critical for

its function during axon regrowth.

Tsr promotes branching of adult γ-neurons

While examining tsrN121 SCCs, we noticed that even axons that extended almost

normally, exhibited elevated occurrences of swellings that resemble lamellipodia-like

protrusions. To test whether these might represent failed branching sites, we

quantified branches (> 5µm) and found that tsr mutant axons had significantly fewer

branches than WT axons (Fig 2A-B, D). Consistent with their effect on axon growth,

expression of both UAS-tsr.N and UAS-tsr.S3A within tsr mutant axons rescued the

branching defect, interestingly to a similar extent, while UAS-tsr.S3E did not (Fig 2C-

D and S3).

Taken together, we propose that Tsr promotes axon elongation as well as branching.

Whether these two functions are linked and utilize the same mechanism of action

remains unclear. Interestingly, a recent study suggests that axon branching and

growth of γ-neurons may be linked. The authors propose that branching allows

axons to bypass obstacles more efficiently (Razetti et al., 2018). In addition, a recent

paper demonstrated that the MT regulator Efa6 is also required for both normal

branching and growth, further implying that these two processes may be

mechanistically related, even in cultured neurons, where presumably the

extracellular environment plays a lesser role (Qu et al., 2019). Even in NBCs, tsr

mutant axons predominantly stop at the branchpoint, suggesting that branching is

key to successful growth. This might also be consistent with the lack of growth

defects in α/β neurons due to their much simpler morphology.

Tsr γ-neurons show reduced neurite dynamics

To further probe the effects of Tsr on growth dynamics, we wanted to subject

neurons undergoing growth to time-lapse imaging. Unfortunately, live imaging of

SCCs during remodeling is practically impossible because the process occurs in a

deep pupal structure. Likewise, SCCs are extremely difficult to visualize in the ex

vivo brain culture system that we have devised (Rabinovich et al., 2015). In order to

circumvent this, we turned to primary cultures of sparsely labeled but densely plated

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WT or mutant γ-neurons (Marmor-Kollet and Schuldiner, 2016). Brains containing

WT or tsrN121 MARCM clones were dissociated and plated for 2 days in vitro (DIV)

before subjected to time-lapse imaging (Fig 2E-F). WT neurons exhibited dynamic

extension-retraction (Fig 2E, G; S movie 1), while, in contrast, tsrN121 neurons

exhibited significantly reduced neurite dynamics (Fig 2F-G, S movie 2). Additionally,

tsrN121 neurons grew shorter neurites (Fig 2H) and branched less (Fig 2I), similarly to

the in vivo effects. Additionally, subjection of primary cultures of WT γ-neurons to

Latrunculin (known to promote actin disassembly, Spector et al., 1983) or

Jasplakinolide (known to interfere with disassembly, Cramer, 1999) both resulted in

decreased sprouting and growth in vitro (Fig S3C-F). Together, these data suggest

that precise actin dynamics and the balance between assembly and disassembly are

crucial for normal neurite extension and branching.

Tsr does not promote regrowth by increasing G-actin or barbed ends

availability

To inspect the hypothesis that Tsr promotes regrowth by increasing G-actin levels,

as suggested by the “treadmilling” model (Shekhar and Carlier, 2017), we performed

epistasis experiments in which we aimed to increase the availability of G-actin.

Increasing G-actin availability by overexpressing either act5c (the fly ortholog of β-

actin) or Chic, the fly profilin (which delivers G-actin to actin elongation factors), both

significantly exacerbated the regrowth defect of tsrN121 SCCs, in contrast to our

original prediction based on the aforementioned hypothesis (Fig 3A-D,H, S4A-B).

Based on these results, we wanted to inquire whether profilin and cofilin antagonize

each other in the context of axon regrowth. In a parallel project, we demonstrated

that perturbing Chic (by RNAi or mutant analysis) also results in a severe γ-axon

regrowth defect (Yaniv et al., 2019 under revision; Fig S4C), which can be rescued

by overexpressing UAS-Chic. Accordingly, and again in contrast to our original

prediction, knocking down Chic levels by RNAi within tsr mutant clones ameliorated

growth defects (3E,H), indeed suggesting they counteract each other’s function.

Next, we aimed to examine the hypothesis that Tsr severing might promote growth

by increasing the fraction of barbed ends that are accessible to polymerization. We

thus expressed RNAi transgenes targeting capping protein-a (cpa) and capping

protein-b (cpb) (Ogienko et al., 2013). Inconsistent with the above hypothesis, we

found that knocking down cpa (but not cpb) enhanced the tsrN121 regrowth defect

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(Fig 3F-H, S4D). Given that cpa and cpb are known to function as an obligatory

heterodimer, our results mostly stem from different RNAi efficiency, something we

unfortunately cannot test due to unavailability of antibodies targeting capping

proteins.

Taken together, these results suggest that increasing G-actin concentration (by

overexpressing act5C or Chic) or increasing the availability of barbed ends (by

knockdown of cpa) exacerbated growth defects, while reducing F-actin (by knocking

down Chic) ameliorated it. Unfortunately, the currently available reporters to examine

G/F actin ratio did not result in meaningful results due to high background, likely due

to the dense neuropil structure. Nonetheless, together these data suggest that Tsr

does not promote regrowth by increasing actin polymerization. Instead, since

increasing G-actin further impairs regrowth in Tsr mutants, we suggest that Tsr may

be important for clearing actin polymers.

Microtubule localization is disrupted in tsr mutants

We turned to examine whether Tsr is required for the elimination of actin structures,

thereby allowing MT elongation (Flynn et al., 2012; Tedeschi et al., 2019). First, we

determined that F-actin levels were indeed increased within tsr SCCs compared to

WT (Fig. 4A-C). Next, we co-expressed a membrane bound tandem tomato (mtdT)

and a tubulin reporter (α-tubulin84B.tdEOS), which in WT SCCs, demonstrated that

MTs and membrane markers seem to overlap (Fig 4D). In contrast, MTs do not

seem to extend into the enlarged filopodia-like structures (Fig 4E) in tsr mutants.

Interestingly, quantification of the co-localization (assessed by the Pearson’s

coefficient) revealed that colocalization is decreased in both axon branchpoints and

tips but remains unaffected in the main axon shaft (Fig 4F). These results suggest

that tsr severs F-actin to clear actin aggregates, which is crucial for proper MT

protrusion. To demonstrate this in a more direct manner, we attempted to express

the MT reporter α-tubulin84B.tdEOS in combination with the F-actin reporter F-tractin

but unfortunately due to rapid bleaching were unable to extract meaningful high-

resolution images. Therefore, while our findings support the hypothesis that Tsr

reduces F-actin accumulations to allow MT elongation, further exploration is

necessary. These findings are consistent with in vitro studies demonstrating

increased F-actin levels in ADF/cofilin mutants. Actin accumulations were suggested

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to obstruct MTs and misdirect them, thus causing a decrease in neurite formation

(Flynn et al., 2012).

To conclude, we have delved deeper into the role of Tsr during axon formation,

including regrowth and branching. We have followed up on several in vitro studies

and showed that Tsr regulates growth and branching in vivo, presumably by severing

F-actin thereby allowing the protrusion of MTs into the potential growth cone.

Materials and methods

Generation and imaging MARCM Clones

MARCM clones of MB neurons and single cell clones were generated at NHL and

examined later, as described previously (Lee et al. 2000b). Brains were mounted on

Slowfade (Invitrogen) and imaged on Zeiss LSM800 confocal microscopes.

Immunostaining and imaging:

Drosophila brains were dissected in cold ringer solution, fixed using 4%

paraformaldehyde for 20 min at room temperature (RT), and washed in phosphate

buffer with 0.3% Triton-X (PBT); 3× immediate washes followed by 3 × 20-min

washes. Non-specific staining was blocked by 5% inactivated goat serum in PBT.

Brains were subjected to primary antibody overnight at 4°C. Primary antibodies

included: Chicken anti-GFP (GFP-1020; AVES), 1:500; mouse monoclonal anti-FasII

(1D4), 1:25 (Developmental Studies Hybridoma Bank; DSHB). Brains were washed

with PBT (3× immediate washes and 3 × 20-min washes), and then stained with

secondary antibodies for 2 h at RT. Secondary antibodies included: Alexa fluor 647

goat anti-mouse (A-21236; Invitrogen), 1:300; FITC donkey anti-chicken 1:300 (703-

095-155; Jackson immunoresearch), 1:300. Brains were mounted on Slowfade (S-

36936; Invitrogen) and imaged on Zeiss LSM 800 confocal microscope. Images were

processed with ImageJ (NIH).

Drosophila melanogaster rearing and strains

All fly strains were reared under standard laboratory conditions at 25 °C on

molasses-containing food. Males and females were chosen at random. Unless

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specifically stated otherwise, the relevant developmental stage is adult, which refers

to 3–5 days posteclosion.

tsrN121, tsrN96A, UAS-tsr.N, UAS-tsr.S3A, UAS-tsr.S3E, UAS-act5c, UAS-LifeAct-

Ruby, UAS-F-tractin.tdTomato, UAS-α-tubulin84B-tdEOS and RNAi lines (cpa, cpb,

chic) were all obtained from the Bloomington Drosophila stock center (Indiana

University, USA). UAS-Chic was kindly provided by F. Besse (Medioni et al., 2004).

Drosophila genotypes used in this study are listed in Supplementary Table S1. Sprouting assay

Dissociation and plating of cells has been previously described (Marmor-Kollet and

Schuldiner., 2016). Briefly, L3 brains were dissociated in 10mg/ml collagenase

(Sigma), washed 3 times with PBS and resuspended in Schneider’s medium (Sigma)

supplemented with 10% heat-inactivated fetal bovine serum and antibiotics.

Dissociated cells were plated in glass bottom 96-well plates coated with poly-Lysine

(MatTek) at ~10 brains/well in a volume of ~30µl in culturing media. One hour after

plating, media was added to a total volume of 200µl. Fluorescent cells were imaged

2 days after plating (DIV) and neurite length was calculated using the Simple Neurite

Tracer plugin in Fiji. 10µM LatrunculinB or 10µM Jasplakinolide (Life Technologies)

were added 1DIV and incubated an additional 24h before imaging.

Sprouting live imaging was performed on cultured neurons labelled with CD8:GFP

and Ftractin-tdT starting at 48h in vitro (2DIV). They were imaged over a period of 10

hours, at 15min intervals. For analysis of dynamics, total neurite length was

measured at every 15min interval (using Simple Neurite Tracer) for a period of 3

hours. Changes between time points were summed for each 1h period and averaged

over 3 hours.

Statistical analysis

We strove for an n>10 brain hemispheres, n’s are shown in the quantifications within

the figures. In the sprouting experiments, n represents cells. No samples were

excluded from the analyses.

For the quantification of developmental regrowth in MARCM clones (Figure 1M), we

used a method previously described (Yaniv et al., 2012). In short, we determined the

γ lobe occupancy by comparing the clonal (GFP) vs non clonal (FasII staining) in the

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Z-plane cross section. To calculate the regrowth index, we then divided the lobe

occupancy of the clonal axons at a distal section by a proximal section.

For analysis of single cell clones (Fig 1N, 3H), the furthest point of innervation into

adult γ lobe was measured on the confocal Z stack and divided by the total length of

the γ lobe (determined by FasII staining). See Fig S1F for details.

For in vivo branching (Figure 2D) the amount of secondary branches > 5µm were

counted and normalized to 100µm of axon length.

For LifeAct-Ruby intensity (Fig 4C): in each image the intensity of both LifeAct-Ruby

and GFP were measured at two different points along the axon as well as at the

axon tip and normalized to the background.

For MT and membrane bound RFP co-localization (Fig 4F) in each neuron at least

two branch points, two axon terminals (if available) and two points along the main

axon were measured for both GFP and RFP. Co-localization, represented by

Pearson’s coefficient, was measured using the Coloc2 Fiji plugin.

Statistical analysis was performed by a one-way ANOVA including all groups

followed by a Tukey post-hoc test (Fig 1M-N, 3H) or student’s T-test (Fig 2G-I, 4C,F).

Acknowledgments

We thank F. Besse and the Bloomington Stock Center for reagents; monoclonal

antibodies were obtained from the Developmental Studies Hybridoma Bank

developed under the auspices of the NICHD and maintained by the University of

Iowa. This work was supported by the European Research Council (erc),

consolidator grant “AxonGrowth”. Fly food for this project was funded by the Women

Health Research Center. O.S. is the Incumbent of the Prof. Erwin Netter Professorial

Chair of Cell Biology.

Competing interests

No competing interests declared

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Figures

Figure 1: Tsr is required for axon growth of MB γ neurons

(A) Schematic representation of γ-neuron developmental remodeling. den-dendrites;

p-peduncle; d-dorsal lobe; m-medial lobe; APF – after puparium formation.

(B-L) Confocal Z-projections of WT (B, E, H, J), tsrN121 (C, F, I, K) or tsrN121

additionally expressing UAS-tsr.N (D, G, L) in γ-neuron MARCM NB (B-G) or SC (H-

I) clones. Asterisks mark γ-lobe edge.

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(M) Boxplot quantification of γ-axon regrowth, depicted as a regrowth index. See

Yaniv et al (2012) for quantification method.

Here and in all boxplots: boxes encompass the values in between the 1st – 3rd

quartiles; whiskers are ± 1.5 IQR; median (line), mean (blue triangle) and outliers

(red diamond). ***p<0.005 **p<0.01 *p<0.05

Grey is OK107-Gal4 (B-D) or R71G10-Gal4 (H-I) driven mCD8::GFP. Green is

R71G10-Gal4 (E-G) driven mCD8::GFP. Magenta represents FasII staining.

Scale bar is 20µm in all images, unless otherwise mentioned.

(N) Boxplot quantification of phenotypic severity shown as ratio of single cell clone

(scc) length to entire lobe length.

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Figure 2: Tsr is required for γ-neuron branch dynamics

(A-C) Confocal Z-projections of WT (A), tsrN121 (B) or tsrN121 additionally expressing

UAS-tsr.N (C) γ-neuron MARCM SCCs. Bottom panels are tracings of the primary

axon in red and secondary branches in cyan. Arrowheads point to axonal structures

that appear to be failed branch points.

Grey is R71G10-Gal4 driven mCD8::GFP

(D) Boxplot quantification of secondary branches (>5µm) per 100µm of primary

axon.

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(E-F) Time-lapse series (15min increments) of confocal Z-projections of WT (E) or

tsrN121 (F) primary γ-neurons derived from L3 brains. White and yellow arrows point

to extending or retracting branch, respectively.

(G) Boxplot quantification of cell dynamics expressed as change in neurite length

divided into extensions and retractions per 1h imaging averaged over 3h.

(H) Boxplot quantifications of total neurite length per cell

(I) Boxplot quantification of number of branches per cell

Grey is R71G10-Gal4 driven mCD8::GFP

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Figure 3: Tsr does not promote regrowth by increasing G-actin levels or by

uncapping

(A-G) Confocal Z-projections of WT (A), tsrN121 (B) or tsrN121 additionally expressing

UAS-act5c (C), UAS-Chic (D), chic RNAi (E), cpa RNAi (F) or cpb RNAi (G) in γ-

neuron MARCM SCCs. Dashed line represents the average ratio of tsrN121

SCC/lobe, which is 0.4.

(H) Boxplot quantification of phenotypic severity shown as ratio of single cell clone

(scc) length to entire lobe length.

Green is R71G10-Gal4 driven mCD8::GFP. Magenta represents FasII staining.

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Figure 4: Microtubule localization is perturbed in tsr mutants

(A-B) Confocal Z-projections of WT (A) or tsrN121 (B) adult γ-neuron MARCM SCCs.

Yellow dashed square demarks representative regions used for quantification of

LifeAct intensity in axon shaft and tip.

Green and cyan are R71G10-Gal4 driven mCD8::GFP or LifeAct-Ruby, respectively.

Grey is R71G10-Gal4 driven mCD8::GFP (A1-B1) or LifeAct-Ruby (A2-B2). Magenta

is FasII staining.

(C) Boxplot quantification LifeAct to CD8::GFP intensity ratio in marked regions in A,

B.

(D-E) Confocal Z-projections of WT (D) or tsrN121 (E) adult γ-neuron MARCM SCCs.

Insets correspond to dashed boxes as examples for regions used for colocalization

analyses – in this case, an axon tip.

Magenta and green are R71G10-Gal4 driven mtdT or α-tubulin84B-tdEOS,

respectively. Grey is R71G10-Gal4 driven mtdT (D1-E1) or α-tubulin84B-tdEOS (D2-

E2). Scale bar is 10µm

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(F) Boxplot quantification of mtdT (membranes) and α-tubulin84B-tdEOS (MT)

colocalization. In each neuron at least one axon tip, branch point, and axon shaft

were quantified. Colocalization was tested using Pearson’s coefficient. While in

tsrN121 there is less colocalization in both the axon tip and branch point, there is not

difference in the main axon shaft.

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WT

Adu

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G H I JFtsrN121

FasIIOK107>CD8

γ lobe length

γ scc length

ratio=γ scc length/γ lobe length

tsrN

96A

B

E

Adult γ neuronsFigure S1

FasII71G10>CD8

min max

Msp300Gene M

ax e

xpre

ssio

n

corashisalsMicalAnxB11CG9426FhosDAAMmimGelchicdiaCG45186CG15097CG6891Abp1FrlsnHsp23scraGMFjubpod1FimcibenaArpc4tsrzipflrcpbcaptArpc3Aalpha-Spectmodbtbeta-SpecdidumDysCLIP-190jarVrp1spirArpc2

L2 L3 0 3 6 12 18 21 24 27 30 adult159

Pupa (hrs after pupation)

min

max

A

Adu

lt α/

β ne

uron

s

FasIIOK107>CD8

DCtsrN96AtsrN121WT

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FigureS1:Expressionpatternofactinrelatedgenes,phenotypeoftsrN96Aandphenotypesoftsrinɑ/βneurons(A)HeatmapdepictingtherelativeRNAexpressionlevelsofgenesrelevanttoactindynamicsthroughoutdevelopmentfrom2ndinstarlarva(L2)toadult.Thepurplescaledepictsthepeakexpressionofeachgenerelativetootherspresentedinthescheme.Genesareorderedbasedontheirclusteredexpression-seeAlyagoretal(2018)fortechnicaldetails.(B)Confocalz-projectionsofadulttsrN96AMBγneuronMARCMneuroblastclones.(C-E)ConfocalZprojectionsofWT(C),tsrN121(D)ortsrN96A(E)inadultMARCMneuroblastclonesofα/βneurons.(F)Schemedepictingthequantificationofsinglecellclonephenotypeseverity:IneachZprojection,therelativelengthofthelabelledsinglecelloutoftheentirelobewascalculated.(G-J)Confocalz-projectionsofadultMBγneuronMARCMsinglecellclonesinWT(G)ortsrN121displayingohenotypicvariability(H-J).GreenisR71G10-Gal4(B)orOK107-Gal4(C-D,G-J)drivenmCD8::GFP.MagentaisFasIIstaining.Scalebaris20µm.

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FigureS2:AxongrowthrescueexperimentsdemonstratetheimportanceofphosphoregulationofTsr(A-B)ConfocalZprojectionsofγneuronMARCMsinglecellclonesoftsrN121additionallyexpressingUAS-tsr.S3A(A)orUAS-tsr.S3A(B).QuantificationshownininFig1N.GreenisR71G10-Gal4drivenmCD8::GFP.MagentaisFasIIstaining

+UAS-tsr.S3A +UAS-tsr.S3E

Adu

lt

A B

tsrN121Figure S2

FasII71G10>CD8

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FigureS3:Branchingrescueexperimentsinvivo,andpharmacologicalmanipulationsofsproutingneurons(A-B)ConfocalZprojectionsofγneuronMARCMsinglecellclonesoftsrN121additionallyexpressingUAS-tsr.S3A(A)orUAS-tsr.S3A(B).Toppanelsfocusontheγaxonregion,bottompanelissameimagewiththeprimaryaxonlabeledinredandsecondarybrancheslabeledincyan.Arrowheadspointtostructureswithintheaxonthatappeartobefailedbranchpoints.GreyisR71G10-Gal4drivenmCD8::GFP.Scalebaris20µm(C-E)ConfocalZprojectionsorprimaryγneuronsderivedfromL3brainsuntreated(C),ortreatedwith10µMJasplakinodinefor24h(D)or10µMLatrunculinBfor24h(E).(F)BoxplotquantificationsofC-E:totalneuritelengthpercell.

Figure S3

Untreated

2DIV

+10µM Jasplakinolide +10µM LatrunculinB

50

100

150

200

250

Tota

l neu

rite

leng

th (μ

m)

Untreated

(n=14) 10µM

Jasplakinolide

(n=13) 10µM

LatrunculinB

(n=26)

***

***

24h treatment

C D EF

tsrN121

+UAS-tsr.S3E+UAS-tsr.S3AA

A’

B

B’Adu

lt 71G10>CD8

1º branch2º branch

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FigureS4:ControlexperimentsofactinandcappingproteinperturbationsinTsrWTanimals(A-D)ConfocalZprojectionsofMBγneuronsexpressingUAS-act5c(A),UAS-Chic(B),chicRNAi(C),cpbRNAi(D).GreenisR71G10-Gal4drivenmCD8::GFP.MagentarepresentsFasIIstaining.Scalebaris20µm

Figure S4

DA B C

WT

FasII71G10>CD8

+cpb RNAi+UAS-act5c +UAS-Chic +chic RNAi

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TableS1.DrosophilaGenotypesusedinthisstudy

hsFLPisy,w,hsFLP122;CD8isUAS-mCD8::GFP;mtdTisUAS-myristoylatedtdTomato;G13is

FRTon2R;Gal80isTubP-Gal80.Malesandfemaleswereusedinterchangeablybutonlythe

femalegenotypeismentioned.

Figure Panels Detailedgenotype

Figure1 B,E,H,J hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13

C,F,I,K hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121

D,G,L hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-tsr.N/+

Figure2 A hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13

B hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121

C hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-tsr.N/+

Figure3 A hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13

B hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121

C hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-act5c/+

D hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-chic/+

E hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-chicRNAi/+

F hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-cpaRNAi/+

G hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-cpbRNAi/+

Figure4 A hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13;UAS-LifeAct-Ruby/+

B hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-LifeAct-Ruby/+

D hsFlp,mtdT/+;71G10-Gal4,G13,Gal80/G13;UAS-α-tubulin84B-tdEOS/+

E hsFlp,mtdT/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-α-tubulin84B-tdEOS/+

FigureS1 A hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN96A

C hsFlp,CD8/+;G13,Gal80/G13;OK107-Gal4/+

D hsFlp,CD8/+;G13,Gal80/G13,tsrN121;OK107-Gal4/+

E hsFlp,CD8/+;G13,Gal80/G13,tsrN96A;OK107-Gal4/+

G hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13

H-J hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121

FigureS2 A hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-tsr.S3A/+

B hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-tsr.S3E/+

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FigureS3 A hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-tsr.S3A/+

B hsFlp,CD8/+;71G10-Gal4,G13,Gal80/G13,tsrN121;UAS-tsr.S3E/+

FigureS4 A 71G10-Gal4,CD8/+;cpbRNAi/+

B 71G10-Gal4,CD8/+;UAS-act5c/+

C 71G10-Gal4,CD8/+;UAS-Chic/+

D 71G10-Gal4,CD8/+;chicRNAi/+

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Supplementalmovies

Movie1:ConfocalZprojectionsofliveimagingofWTprimarycellsderivedfromL3brainsstartingat2daysinvitro(2DIV).Cellswereimagedoveraperiodof10hours,at15-minuteintervals.Whitearrowsmarkextendingorretractingneurite.GreenisR71G10-Gal4drivenmCD8:GFP.RedisR71G10-Gal4drivenF-tractin.tdTomato.

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Movie2:ConfocalZprojectionsofliveimagingoftsrN121primarycellsderivedfromL3brainsstartingat2DIV.Cellswereimagedoveraperiodof10hours,at15-minuteintervals.Whitearrowsmarkextendingorretractingneurite.GreenisR71G10-Gal4drivenmCD8:GFP.RedisR71G10-Gal4drivenF-tractin.tdTomato.

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