short title: simultaneous recruitment of tpc subunits at ...3 71 associated with a hexameric...
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Short title: Simultaneous recruitment of TPC subunits at the PM 1
The author for contact details is Daniël Van Damme ([email protected]). 2
3
4
High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE 5
complex subunits. 6
7
Jie Wang1,2
, Evelien Mylle1,2
, Alexander Johnson3, Nienke Besbrugge
1,2, Geert De Jaeger
1,2, Jiří Friml
3, 8
Roman Pleskot1,2
, Daniel Van Damme1,2,*
9
10 1Ghent University, Department of Plant Biotechnology and Bioinformatics, Technologiepark 71, 9052 11
Ghent, Belgium 12 2VIB Center for Plant Systems Biology, Technologiepark 71, 9052 Ghent, Belgium 13
3Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria 14
15
One sentence summary: Lowering the temperature increases spatiotemporal resolution of protein 16
recruitment at the plasma membrane. 17
18
Author Contributions: JW, EM, AJ and NB designed and performed experiments and/or generated 19
material. JW, EM, AJ, GDJ, JF, RP and DVD designed experiments, analyzed data and discussed results. 20
JW, RP and DVD wrote the initial draft of the manuscript. AJ, GDJ, JF, RP and DVD contributed to 21
finalizing the paper. 22
23
Abstract 24
The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC contains six 25
evolutionary conserved subunits and two plant specific subunits, AtEH1/Pan1 and AtEH2/Pan1, which are 26
not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of 27
the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell 28
imaging at physiological and a lowered temperature. Our data show that lowering the temperature slows 29
down endocytosis and thereby enhances the temporal resolution of the differential recruitment of 30
endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit 31
TPLATE, and the AtEH/Pan1 proteins, exhibited simultaneous recruitment at the PM. These results, 32
together with our co-localization analysis of different TPC subunits, allow us to conclude that in plant cells, 33
TPC is not recruited to the PM sequentially but as an octameric complex. 34
Key words 35
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Plant endocytosis, live-cell imaging, TPLATE complex, temperature control. 36
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Introduction 38
Clathrin-mediated endocytosis (CME) is the best-studied and predominant endocytic pathway in 39
eukaryotes to internalize plasma membrane (PM) proteins and extracellular materials, commonly termed 40
“cargo”, into cells (Bitsikas et al., 2014; Dhonukshe et al., 2007; Kitakura et al., 2011; Robert et al., 2010). 41
The formation of clathrin-coated vesicles (CCVs) requires several highly coordinated stages: initiation, 42
cargo selection, coat assembly, scission and vesicle uncoating (McMahon and Boucrot, 2011). Though 43
the initiation of CME at the PM remains poorly understood in plants, similarly to other systems, adaptor 44
proteins are presumed to recognize cargo proteins via cargo-recognition motifs and subsequently recruit 45
clathrin triskelia through clathrin-binding motifs (Zhang et al., 2015). As clathrin does not bind directly to 46
the PM nor to the cargo proteins, adaptor proteins thus play an essential role to link the PM and the 47
clathrin cage (McMahon and Boucrot, 2011). 48
Two early-arriving adaptor protein complexes function at the PM in plants; the heterotetrameric Adaptor 49
Protein-2 complex (AP-2) and the octameric TPLATE complex (TPC). AP-2 comprises of two large (AP2A 50
and AP2B or α and β), one medium (AP2M or μ) and one small subunit (AP2S or σ) (Di Rubbo et al., 51
2013). TPC contains eight components; evolutionary conserved TPLATE, TML, TASH3, LOLITA, TWD40-52
1, TWD40-2, and plant-specific AtEH1/Pan1 and AtEH2/Pan1 subunits (Gadeyne et al., 2014; Hirst et al., 53
2014). AP-2 function is important for somatic plant development as single Arabidopsis ap-2 mutants 54
display developmental defects, yet still result in viable plants (Di Rubbo et al., 2013; Fan et al., 2013; Kim 55
et al., 2013; Yamaoka et al., 2013). The TPC is essential for both pollen and somatic plant development 56
as knockout, or knockdown, of single subunits of TPC in Arabidopsis leads to pollen or seedling lethality 57
respectively (Gadeyne et al., 2014; Van Damme et al., 2006). 58
TPC is an evolutionary ancient protein complex that has been so far experimentally characterized only in 59
plants and in an amoeba, Dictyostelium discoideum (Gadeyne et al., 2014; Hirst et al., 2014). TPC in 60
plants was identified as an octameric complex by tandem affinity purification (TAP) experiments (Gadeyne 61
et al., 2014). Recently, we also identified an important role for the AtEH/Pan1 proteins in actin-regulated 62
autophagy and in recruiting several components of the endocytic machinery to the AtEH/Pan1 positive 63
autophagosomes (Gadeyne et al., 2014; Wang et al., 2019). TPC in Dictyostelium (described as TSET) 64
was however identified as a hexameric complex lacking the AtEH/Pan1 homologs (Hirst et al., 2014). 65
Although Amoebozoa contain homologuous proteins to AtEH/Pan1, these resemble more closely Ede1 66
than Pan1 (Gadeyne et al., 2014; Wang et al., 2019). 67
Truncation of the TML subunit of TPC forces the complex into the cytoplasm and this correlates with the 68
dissociation of AtEH/Pan1 proteins from the complex (Gadeyne et al., 2014). It therefore appears that 69
TPC composition depends on its localization and that the two AtEH/Pan1 subunits might be peripherally 70
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associated with a hexameric subcomplex of TPC, which would resemble to TSET in Dictyostelium. To 71
investigate whether there is a differential order of recruitment between both AtEH/Pan1 proteins and the 72
remaining hexameric subcomplex, we performed dual color time-lapse microscopy of CME in etiolated 73
Arabidopsis epidermal hypocotyl cells. We also took advantage of lowering the temperature of our 74
samples to slow down CME and thereby increase the spatiotemporal resolution. Altogether, our data 75
strongly suggest that TPC is recruited as the octameric complex to the PM, where it functions as the early 76
adaptor complex for plant CME. 77
78
Results 79
Lowering the experimental temperature reduces CME kinetics 80
TPC is proposed to serve as an early adaptor complex (Gadeyne et al., 2014), however temporal 81
resolution however remains the biggest challenge to monitor the dynamic recruitment of different 82
endocytic protein players (Bashline et al., 2015; Fan et al., 2013; Fujimoto et al., 2010; Gadeyne et al., 83
2014). As lowering the temperature generally slows down dynamics of cellular processes (Das et al., 84
1966), we hypothesized that by lowering the temperature, we could also slow down dynamics of endocytic 85
events and therefore enhance the spatiotemporal visualization of the differential recruitment of the 86
endocytic players at the PM. 87
To time how intracellular dynamics respond to decreasing the temperature in plant cells, we firstly 88
imaged Arabidopsis plants expressing the microtubule (MT) binding protein EB1a-GFP (Van Damme et al., 89
2004). Hypocotyl cells of Arabidopsis seedlings expressing EB1a-GFP were imaged at 20 °C for 5 min, 90
then the temperature was lowered to 10 °C with the aid of the CherryTemp Heater Cooler system and the 91
effect on microtubule polymerization rate was assessed. Lowering the temperature had an immediate 92
effect on the MT polymerization speed, measured as EB1a labeled tracks in time-projection images (Fig. 93
S1 A). Visualization and quantification of the MT growth dynamics further confirmed that the temperature 94
shift was transmitted to the seedling almost instantaneously (Fig. S1). 95
To examine the capacity of lowering the temperature to slow down endocytosis, etiolated hypocotyl cells 96
expressing endocytic markers CLC2-mKO and TPLATE-GFP the temperature was shifted during image 97
acquisition and no obvious defect in the recruitment was observed. Density analysis of early-arriving 98
(TPLATE and TML) as well as late arriving DRP1A furthermore showed that lowering the temperature did 99
not visually affect the PM recruitment of these endocytic markers while it prolonged their lifetime at the PM 100
instantly (Figure 1 and Figure 2A). 101
To further evaluate how lowering the temperature affects the dynamic behavior of endocytic proteins, we 102
carefully measured the dynamics of TPLATE, TML and DRP1a at different temperatures. Kymograph and 103
histogram analysis of measured lifetimes confirmed that lowering the temperature from 25 to 10 °C 104
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correlated with a gradual increase in lifetime of endocytic proteins at the PM (Fig. S2). For TPLATE and 105
TML, we observed a strong increase in average lifetime between 15 and 12 °C. Therefore, we selected 106
25 °C and 12 °C as the two temperatures for our future experiments. 107
Visualizing the individual lifetimes of the three markers at both temperatures in a histogram showed a 108
clear shift of the distribution to longer lifetimes (Fig. 2B) and this correlated with a significant reduction in 109
the internalization of the styryl dye FM4-64, which is used as a proxy for endocytic flux (Bashline et al., 110
2013; Dejonghe et al., 2016; Dejonghe et al., 2019; Fan et al., 2013; Gadeyne et al., 2014; Van Damme et 111
al., 2011) (Fig. 2C). Furthermore, photo-bleaching experiments showed a dramatic reduction in the 112
recovery of endocytic foci at PM at the low temperature, in agreement with reduced dynamics of the 113
process (Fig. S3). These results together, show that lowering the temperature in etiolated seedlings using 114
our experimental setup slows down CME efficiently and rapidly. 115
116
Lowering the temperature enhances the temporal resolution of plasma membrane recruitment. 117
Having established the effect of lowering the temperature on endocytic dynamics in Arabidopsis cells, we 118
then examined whether we would be able to enhance the temporal difference between an early (TPLATE) 119
player and a late one (DRP1A). We therefore generated a dual marker line of TPLATE-GFP (Van Damme 120
et al., 2006) and DRP1a-mRFP (Mravec et al., 2011) and compared their temporal behavior at both 121
temperatures. When imaged at 25 °C, the time-lapse images and kymographs showed multiple 122
independent events where TPLATE-GFP foci clearly appeared earlier than DRP1a-mRFP at the PM, while 123
they disappeared together (Fig. 3A, B and Fig. S4). Lowering the temperature prolonged the lifetime of 124
both TPLATE-GFP and DRP1a-mRFP (Fig. 3A-B), whereas their departure remained synchronized. 125
Overall, the temporal difference between their PM recruitment was dramatically enhanced (Fig. 3A, B and 126
Fig. S4), which resulted in a very significant mean difference of the paired lifetimes and the large Hedge’s 127
g value, which indicates the effect size (Fig. 3C). 128
We performed a similar experiment comparing TPLATE and CLC2, which were previously shown only to 129
have minor differences in their PM recruitment (Gadeyne et al., 2014; Narasimhan et al., 2020). Here, we 130
also observed a temperature-enhancing effect of their differential recruitment with TPLATE being recruited 131
before CLC2, yet less pronounced than the difference between TPLATE and DRP1a (Fig. 3D to F and Fig. 132
S4). 133
Taken together, these results show that lowering the temperature slows down dynamics of endocytosis 134
as well as enhances the spatiotemporal resolution of recruitment of the endocytic proteins at the PM. 135
TPLATE is closely associated with the AtEH/Pan1 proteins at the plasma membrane 136
The absence of AtEH1/Pan1 and AtEH2/Pan1 as subunits of TSET (Hirst et al., 2014), together with the 137
observation that inducing mislocalization of TPC to the cytoplasm leads to loss of those two subunits from 138
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the complex (Gadeyne et al., 2014) and the observation that AtEH/Pan1 proteins have a specific role in 139
promoting autophagy (Wang et al., 2019), suggest that TPC could be in essence a hexameric complex, 140
which temporarily gains two additional subunits. To reveal the spatiotemporal relationship among the TPC 141
subunits, we visualized their dynamic behavior at the PM using multiple dual-color labeled lines. To 142
evaluate the functional association of TPC subunits at PM, we crossed complemented mutant lines of core 143
subunits (TPLATE, TML and TWD40-1) and generated double complemented homozygous mutant lines. 144
We also combined complemented mutants of the core subunit TPLATE with AtEH1/Pan1 or AtEH2/Pan1 145
and generated the respective double complemented, double homozygous mutant lines. As a first 146
indication for differential recruitment, we monitored the steady-state percentage of co-localization between 147
the markers using a flattened projection of five consecutive frames. Comparing an early endocytosis 148
marker such as TPLATE, with a late marker such as DRP1A revealed that the differential arrival between 149
those makers leads to a substantial non-colocalizing fraction (roughly 40%, Fig. 4A). 150
Combining TPLATE-TagRFP with TML-GFP resulted in around 77% of total foci where TML and 151
TPLATE overlapped (Fig. 4B). We also tested functional association when combining TPLATE-GFP with a 152
TWD40-1-mRuby3 complemented line (Fig. S5 and Table 1). Similarly, as for TML and TPLATE, 153
combining TPLATE-GFP and TWD40-1-mRuby3 resulted in a very high percentage of colocalizing foci 154
(Fig. 4C), confirming their intrinsic behavior as part of the same complex. 155
In Arabidopsis seedlings, AtEH1/Pan1 and AtEH2/Pan1 have so far been shown functionally associated 156
with TPLATE at autophagosomes and to be delivered to the vacuole after carbon starvation and Conc A 157
treatment (Wang et al., 2019). To address the recruitment of AtEH/Pan1 and TPLATE in endocytic foci at 158
the PM, we combined AtEH/Pan1 with TPLATE in their respective complemented mutant backgrounds. 159
Similar to the combination of the core subunits, both AtEH1/Pan1-mRuby3 and AtEH2/Pan1-mRuby3 160
showed a very high degree of colocalization with TPLATE-GFP at the PM (Fig. 4D and E). To exclude the 161
possibility that TPLATE and AtEH/Pan1 foci overlapped due to the high density of endocytic foci, we 162
compared the colocalization percentage between TPLATE and AtEH1/Pan1 on images where the 163
TPLATE channel was rotated 90 degrees. This rotation resulted in only around 10% of foci colocalizing 164
(Fig. 5F), confirming that the observed high degree of colocalization is not random. These results also 165
suggest a tight association between TPLATE and the AtEH/Pan1 proteins at the PM. 166
167
AtEHs/Pan1 and the core TPC subunits are simultaneously recruited to the PM 168
To further investigate TPC assembly at the PM at the level of the different subunits, we compared the 169
recruitment and disassociation behavior among pairs of TPC subunits at 25 and 12 °C. As a benchmark, 170
we compared the behavior of two closely related TPC subunits, TPLATE and TML, which by homology to 171
other adaptor complexes are presumed to be part of the core of TPC. 172
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Time-lapse imaging and kymograph analyses showed that dual-labeled TML-GFP and TPLATE-TagRFP 173
foci appear and disappear simultaneously at the PM when imaged at 25 °C (Figure 5A, 5B and S6). 174
Lowering the temperature maintained this simultaneous appearing and disappearing behavior. However, 175
there were very small changes in average lifetime, which were found to be negligible by their effect size 176
(expressed as the Hedge’s g value). These small changes could be explained by there being more signal 177
noise and differential bleaching effects of the fluorophores during the image acquisition at the lower 178
temperature conditions (Figure 5A-5C and S6). 179
We next compared the PM appearance and disappearance behavior of AtEH/Pan1 and TPLATE. 180
Similar to the recruitment behavior of TPLATE and TML foci, when imaged at 25 °C, both AtEH1/Pan1 181
and AtEH2/Pan1 foci simultaneously appeared and disappeared with TPLATE at the PM (Fig.D-E, G-H 182
and S7). Lowering the temperature did not alter the simultaneous recruitment of TPLATE and AtEH/Pan1, 183
and the respective Hedge’s value remained very low (Fig.D-I and S7). These results, together with our 184
colocalization analysis, strongly suggest that AtEH/Pan1 subunits are not individually recruited to, or 185
maintained, at the PM. 186
Discussion 187
Clathrin-mediated endocytosis is a highly dynamic process. It is best understood in mammalian and yeast 188
model systems while little is known in plants. Although very fast endocytic events were observed in 189
neurons (Balaji and Ryan, 2007), in yeast or cultured animal cells, the whole endocytic process takes up 190
to minutes and the physiological role and precise temporal dynamics of endocytic proteins are well defined 191
(Lu et al., 2016; Ma et al., 2016; Pascolutti et al., 2019; Picco and Kaksonen, 2018; Taylor et al., 2011; 192
Wrobel et al., 2019). In plant cells, the endocytic process is much more dynamic, with larger clathrin cages 193
being formed than those in yeast in shorter time periods (Narasimhan et al., 2020), which brings about 194
more technical difficulties in monitoring the temporal recruitment of the diverse endocytic players (Zhang 195
et al., 2015). To better understand the dynamics of endocytosis in plant cells, new approaches are 196
required, which should provide us with enhanced temporal resolution. 197
Here, we report that lowering the temperature using a microfluidics-based on-slide approach can be used 198
as a rapid and efficient tool to slow down intracellular dynamic processes in plant cells similar to earlier 199
reports demonstrating temperature effects on endocytosis and synaptic vesicle recycling in animal cells 200
(Bui and Glavinovic, 2014; Pyott and Rosenmund, 2002). The effect on microtubule growth rate was 201
immediate and the dampening effect on the dynamic of endocytosis, as measured by the dwell time at the 202
PM of several markers, FM-uptake as well as measuring recruitment by FRAP correlated with the 203
reduction in temperature. Lowering the temperature however had no significant effect on the density of 204
endocytic foci, suggesting that cargo proteins could still be adequately recognized. 205
Our data also show that the temporal resolution of the differential recruitment of endocytic players could 206
be enhanced at lower temperatures, which was most apparent between early and late arriving endocytic 207
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proteins. Lowering the temperature also enabled us to slightly enhance the temporal difference between 208
two early arriving proteins, TPLATE and CLC2, indicating that our approach worked. Given the 209
temperature effect on the dynamics of plant processes, awareness should be increased to perform live-210
cell imaging in plants under accurate temperature control. No doubt this will substantially increase the 211
robustness and reproducibility of future image data collection. 212
The TPC is an evolutionary ancient adaptor complex, which was not retained in yeast or animal cells (Hirst 213
et al., 2014). It is proposed to function as an adaptor complex during CME and has been shown to be 214
recruited to the PM earlier than Dynamin-related proteins while only slightly earlier than clathrin(Gadeyne 215
et al., 2014; Narasimhan et al., 2020). Dictyostelium discoideum contains a similar complex, which 216
however lacks the AtEH/Pan1 proteins and in contrast to TPC, TSET is not essential (Gadeyne et al., 217
2014; Hirst et al., 2014). These AtEH/Pan1 proteins both share their male sterility phenotype with the 218
other TPC subunits (Gadeyne et al., 2014; Van Damme et al., 2006; Wang et al., 2019), they are 219
dynamically recruited to the PM (Gadeyne et al., 2014; Wang et al., 2019), associated with the other TPC 220
subunits as well as AP-2, but not when TPC is forced into the cytoplasm using proteomic manipulations 221
(Gadeyne et al., 2014) and functionally associated with several endocytic players during autophagy (Wang 222
et al., 2019). Here we investigated how these AtEH/Pan1 proteins are recruited to the PM in relation to the 223
other TPC subunits. Our live-cell analysis could not identify any deviation in recruitment dynamics 224
between AtEH/Pan1 and TPLATE, mirroring the results observed between the other core subunits 225
TPLATE and TML. The small differences reported could be attributed to technical issues, including 226
differential bleaching effects of fluorophores. The close association, as well as the simultaneous 227
recruitment between TPLATE and AtEH/Pan1 labeled endocytic foci at the PM, therefore allow us to 228
hypothesize that TPC is recruited to the PM as an octameric complex in plants. 229
In plants, lifetimes of endocytic proteins are measured using time-lapse imaging of cells expressing 230
fluorescent labeled proteins imaged via spinning disk microscopy (Bashline et al., 2013; Bashline et al., 231
2015; Dejonghe et al., 2016; Dejonghe et al., 2019; Gadeyne et al., 2014; Zhou et al., 2018) or total 232
internal reflection fluorescence microscopy (TIRF) or variable angle epifluoresence microscopy (VAEM) 233
(Ito et al., 2012; Johnson and Vert, 2017; Konopka et al., 2008; Konopka and Bednarek, 2008a, b; 234
Narasimhan et al., 2020). Based on earlier work in the animal field (Aguet et al., 2013; Compeer et al., 235
2018; Jaqaman et al., 2008; Pascolutti et al., 2019; Wrobel et al., 2019), automated image analysis tools 236
have recently been employed to analyze CME images unbiasedly and rapidly (Johnson and Vert, 2017; 237
Narasimhan et al., 2020). Accurate detection of endocytic events requires high signal-to-noise ratio (SNR) 238
images, a quality which is determined by multiple factors such as the susceptibility of the fluorophore to 239
bleaching and the expression level of the fluorescent reporter and close-to-endogenous levels of 240
fluorescent labeled endocytic proteins often failed to provide sufficient SNR for accurate lifetime 241
measurements (Aguet et al., 2013). Here, we aimed at visualizing the recruitment dynamics of various 242
TPC subunits at the PM. In order to avoid competition with endogenous subunits and to avoid over-243
expression effects such as induction of autophagy (Wang et al., 2019), we opted to work with double-244
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labeled endocytic proteins in either single or double complemented tpc mutant background rather than use 245
overexpression lines. Although our approach represents the optimal strategy from a biological perspective, 246
our transgenic lines, especially those fused with red fluorescent proteins, and combined with extended 247
imaging times under low temperature conditions failed to generate sufficient SNR images for automatic 248
quantification. Actually, the only combination which yielded acceptable data when automatically quantified 249
was our TPLATE-DRP1A combination, as the DRP1A is actually 35S-driven. 250
We therefore generated kymographs of our dual-color endocytic spinning disc movies and measured 251
the dwell-times of the proteins manually. We aimed at measuring several hundred events per experiment 252
and combined measurements of independent persons to achieve an unbiased assessment of the data. 253
We were unable to identify any recruitment of AtEH/Pan1 proteins at the PM which was independent of 254
another TPC subunit. Our findings therefore show that the TPLATE adaptor protein complex is likely 255
recruited to the PM as an octameric unit, both at normal and at lowered temperature conditions. We can 256
however not exclude the possibility that the differential recruitment between the individual TPC subunits 257
are too dynamic to monitor under our limitating, one-second temporal resolution, conditions. However, due 258
to the fact that we were able to detect differences in the CLC2 and TPLATE dynamics, which are of the 259
order of a few seconds, we hypothesize that TPC is recruited as an octameric unit at the PM during CME. 260
The continuous development of novel microscopy as well as labeling techniques will help to overcome our 261
current limitations of working with low-expressing functional fusions and will allow us to understand how 262
endocytosis in plants is executed at high spatiotemporal resolution. 263
264
Materials and Methods 265
Molecular cloning 266
To yield the expression construct for TWD40-1, entry clones of TWD40-1 without a stop codon in 267
pDONR221(Gadeyne et al., 2014) were combined with pB7m34GW (Karimi et al., 2007), pDONRP4-P1R-268
Histone3p (Ingouff et al., 2017), and pDONRP2-P3R-mRuby3 (Wang et al., 2019) in a triple gateway LR 269
reaction (Invitrogen) to generate pH3::TWD40-1-mRuby3. To yield a red fluorophore-tagged TPLATE, 270
TPLATE without stop in pDONR207 (Van Damme et al., 2006) was combined with the Lat52 promotor in 271
pDONRP4P1R (Van Damme et al., 2006), with TagRFP in pDONRP2P3R (Mylle et al., 2013) and with 272
pH7m34GW (Karimi et al., 2007) in a triple gateway LR reaction (Invitrogen) to generate pLat52::TPLATE-273
TagRFP. 274
275
Plant Material 276
The Arabidopsis lines expressing EB1a-GFP (Van Damme et al., 2004), LAT52p::TPLATE-GFP 277
(Gadeyne et al., 2014; Van Damme et al., 2006), TMLp::TML-GFP (Gadeyne et al., 2014), 35Sp::DRP1a-278
mRFP (Mravec et al., 2011) and 35Sp::CLC2(At2g40060)-mKO (Ito et al., 2012) were previously 279
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described. The dual-color line expressing TPLATE-GFP/CLC2-tagRFP in tplate homozygous background 280
was reported previously (Gadeyne et al., 2014). The dual-marker lines expressing TPLATE-281
GFP/AtEH1/Pan1-mRuby3 or TPLATE-GFP/AtEH2/Pan1-mRuby3 in double homozygous mutant (tplate 282
ateh1/pan1 or tplate ateh2/pan1) background were reported before (Wang et al., 2019). 283
To generate TWD40-1-mRuby3 complemented lines, heterozygous mutants of twd40-1-1 (Gadeyne et 284
al., 2014) were transformed with pH3.3::TWD40-1-mRuby3 by floral dip. The T1 plants were selected for 285
the complementation constructs on 1/2 MS plate supplemented with 10mg/L Basta. Resistant plants were 286
genotyped to identify those with a twd40-1-1 heterozygous mutant background. T2 plants expressing 287
TWD40-1-mRuby3 were tested by genotyping PCR to identify homozygous lines for the twd40-1-1 288
insertion mutations. Genotyping PCR was performed on genomic DNA isolated from rosette leaves. 289
Genotyping primers for twd40-1-1 are described before (Gadeyne et al., 2014). 290
For backcross experiments, the complemented lines of TWD40-1-mRuby3 as well as the heterozygous 291
mutant plants of twd40-1-1 were used as male to cross with Col-0 as female. The transfer of the T-DNA, 292
causal to the functionality of the complementing fusion, was analyzed by genotyping PCR on F1 plants. 293
To generate dual-marker lines of TPLATE core subunits, a complemented tplate lines expressing 294
pLat52::TPLATE-tagRFP was generated by dipping tplate(+/-) heterozygous plants with pLat52::TPLATE-295
TagRFP and selecting for the transformants. Homozygous tplate(-/-) plants were identified in the next 296
generations by genotyping PCR. 297
Homozygous tplate mutant plants carrying pLat52::TPLATE-TagRFP were crossed with the 298
complemented tml-1 line expressing TMLp::TML-GFP (Gadeyne et al., 2014). The complemented twd40-299
1-1 lines expressing TWD40-1-mRuby3 were crossed with the complemented tplate line Lat52p::TPLATE-300
GFP, respectively. F2 plants in double homozygous mutant background (tml-1/tplate or tplate/twd40-1-1 301
were identified by genotyping PCR. For the dual-color TPLATE-GFP/DRP1a-mRFP lines, the 302
complemented tplate lines expressing Lat52p::TPLATE-GFP were crossed with 35Sp::DRP1a-mRFP 303
expressing lines and F2 plants in tplate homozygous background were identified by genotyping PCR. 304
Genotyping primers for tplate, tml-1 and twd40-1-1 are described before (Gadeyne et al., 2014). 305
Temperature modulation using the CherryTemp System 306
The CherryTemp system (Cherrybiotech), which enables ultra-fast temperature shifts between 5 °C and 307
45°C was used to modulate and maintain the temperature during the spinning disk imaging 308
(https://www.cherrybiotech.com/heater-cooler-for-microscopy). Prior to imaging, a single etiolated seedling 309
was laid between two coverslips with 1/2 strength MS liquid medium and incubated with the CherryTemp 310
Heater Cooler device for five minutes prior to imaging to stabilize the temperature of the seedling 311
(https://www.cherrybiotech.com/heater-cooler-for-microscopy/temperature-control-for-plant-312
microscopy). 313
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FM4-64 Uptake 314
Prior to imaging, whole 6-day-old Col_0 seedlings were incubated with 4 μM FM4-64 (Invitrogen) solution 315
in 1/2 strength MS liquid medium between 2 coverslips at 25 °C or 12 °C for 30 minutes. 316
Live-cell Imaging 317
A Nikon Ti microscope with the Perfect Focus System (PFSIII) for Z-drift compensation, equipped with an 318
Ultraview spinning-disk system (PerkinElmer) and two 512x512 Hamamatsu ImagEM C9100-13 EMccd 319
cameras was used to image endocytic dynamics. Images of hypocotyl epidermal cells of 4-day old 320
etiolated seedlings expressing single or dual-color fluorophore labeled proteins were acquired with a 100x 321
oil immersion objective (Plan Apo, NA = 1.45). During imaging, the CherryTemp system was used to 322
maintain the temperature of the samples constant. 323
Seedlings expressing GFP fused proteins were imaged with 488nm excitation light and an emission 324
window between 500 nm and 530 nm in single camera mode, or 500 to 550 nm in dual camera mode. 325
Seedlings expressing mKO, mRFP and tagRFP labled proteins were imaged with 561 nm excitation light 326
and an emission window between 570nm and 625nm in single camera mode or 580 to 630 nm in dual 327
camera mode. Single-marker line movies were acquired with an exposure time of 500 ms/frame. Movies 328
of 2 minutes in total were made. Dual-color lines were acquired either sequentially (one camera mode) or 329
simultaneously (two camera mode) with an exposure time of 500 ms/frame. Single camera mode was 330
used for density, colocalization (TML-TPLATE, TPLATE-DRP1A) and life-time (TML-DR1AP,TML-331
TPLATE 25°C) measurements. Dual camera mode was used for colocalization (TPLATE-AtEH1/Pan1, 332
TPLATE-AtEH2/Pan1, TPLATE-TWD40-1, TPLATE-TWD40-2) and lifetime (TPLATE-AtEH1/Pan1, 333
TPLATE-AtEH2/Pan1 and TML-TPLATE 12°C) measurements. For photo-bleaching experiments, 334
seedlings were exposed to 100% power of laser for around 2s during the imaging, using the Photokinesis 335
unit of the PE spinning disk system. 336
Root meristematic epidermal cells of 6-day-old seedlings were acquired with a Zeiss 710 inverted 337
confocal microscope with the ZEN 2009 software package and equipped with a C-Apochromat 40x water 338
Korr M27objective (NA 1.2). FM4-64 was visualized using 561 nm laser excitation and a 650–750 nm 339
detection window. 340
341
Quantification of measurements 342
Dynamics of EB1a-GFP were analyzed using ImageJ equipped with the Trackmate (v4.0.1) 343
plugin(Tinevez et al., 2017). We used the LoG detector with an estimated blob diameter of 10 pixels, a 344
threshold value of 1, using the median filter and sub-pixel localization. An auto initial thresholding, followed 345
by a Linear motion LAP tracker with as parameters a 5 pixel initial search radius, a 5 pixel search radius 346
and a 2 frames maximum frame gap correction, were applied. Tracks with a duration of less than 10 347
frames were excluded and the obtained median speed per track was converted to µm/min using the pixel 348
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size values. Outliers for the median speed were defined by the iterative Median Absolute Deviation 349
Method (MAD) (Leys et al., 2013) and their values were excluded. Three movies coming from two different 350
seedlings were analyzed. 351
The density of labeled endocytic markers was measured in Matlab 2017b using the detection and 352
tracking parts of the of the cmeAnalysis package and further processed as described in (Johnson and 353
Vert, 2017; Narasimhan et al., 2020). Density calculations were obtained from all the tracks within the 354
region of interest (ROI) over certain frames of the movies, which used to produce an average density. The 355
ROI was selected on the middle of the image. The middle frame was used as a reference and the 356
temporal range is based on the middle frame. We used the pixel size and the area to convert this to spots 357
per µm2. For each of the analyzed sample set, eight cells from eight different seedlings were analyzed. 358
359
Objects based co-localization was performed using the ImageJ plugin Distance Analysis (DiAna) (Gilles 360
et al., 2017). Prior to analyzing with DiAna, images were processed with ImageJ. Each channel was 361
processed with a Walking Average of 4 and then merged (also rotated if required). Region of interest 362
were selected based on that they excluded the border of the cells and still represented a good number of 363
objects. Z-projection images were generated of five slices with average intensity. Each channel of Z-364
projected images was processed using Morphological filters from the MorphoLibJ plugin (Legland et al., 365
2016), using the parameters white top-hat, disk element and a 2 pixels radius. A first step in the DiAna 366
plugin is to segment the objects for each channel, which is done by selecting the 3D Spot segmentation 367
tool of the DiAna-Segment plugin. If requested, adapt the calibration by changing the pixel size to 368
1.00001 for all dimensions. Both the noise and seed threshold value were obtained by averaging the 369
maximum intensity of three regions covering only background signal. The spot was defined with a 370
minimum value of 4 and maximum value of 36 pixels. The option to exclude objects on XY edges was 371
activated. Default values were used for the other parameters. Results for number of total objects (Tot) or 372
touching objects (Tou) in image A/B obtained from Diana were recorded. The colocalization ratio of 373
objects was calculated as follows: 374
only (A) % = (Tot A- Tou A)/((TouA + TouB)/2+ (Tot A- Tou A) + (Tot B- Tou B)) * 100 375
only (B) % = (Tot B- Tou B)/((TouA + TouB)/2+ (Tot A- Tou A) + (Tot B- Tou B)) * 100 376
Colocalization % = 100% - only (A)% - only (B)% 377
As a control, one of the channels was rotated 90°C (no interpolation) and analyzed similarly as described 378
above. For each of the analyzed sample set, a minimum of six cells coming from three different seedlings 379
were analyzed. 380
Lifetimes of individual endocytic events were measured from kymographs generated by the Volocity 381
software package (PerkinElmer). To measure paired lifetimes of dual-color kymographs, individual events 382
showing good SNR (signal-noise ratio) in both channels were marked. Following the measurement of the 383
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lifetimes of the marker in the red channel, the lifetime of the marker in the green channel was analyzed. 384
Data was further analyzed in Excel by checking the start position of X from each line of the kymograph to 385
avoid mistakes in pairing the red and green channel values. Calculation to time (ns) of each line was done. 386
Outliers for the life-time differences were defined by the iterative MAD (Leys et al., 2013) and their values 387
were excluded. For each of the analyzed sample sets, minimum 9 movies spread among minimum 3 388
seedlings were analyzed. Except for TPLATE-GFP/CLC2-tagRFP for which 7 movies over 3 seedlings 389
(12°C) and 5 movies over 2 seedlings (25°C) were analyzed. To generate unbiased data, paired lifetimes 390
of endocytic events labeled by dual-color fluorophores were measured by independent persons. 391
392
Statistical analysis 393
The results were analyzed with the estimation method to calculate mean, mean differences, confidence 394
intervals, and Hedges’ g (Claridge-Chang and Assam, 2016; Ho et al., 2019). 95% confidence intervals for 395
the mean differences were calculated using bootstrap methods (re-sampled 5000 times, bias-corrected, 396
and accelerated). Effect size was measured using Hedges’ g and accordingly to the standard practise is 397
referred as ‘negligible’ (g < 0.2), ‘small’ (0.2 < g < 0.5), ‘medium’ (0.5 < g < 0.8) or ‘large’ (g > 0.8) (Cumming, 398
2012). Hedges’ g is a quantitative measurement for the difference between mean indicating how much two 399
groups differ from each other, if Hedges’ g equals 1, the two groups differ by 1 standard deviation. R 400
version 3.6.2 and Rstudio 1.2.5001 were used to calculate the Wilcoxon Signed Rank test (paired 401
samples), the Mann-Whitney U-test and the Welch two sample T-test (Team, 2019). All plots and figures 402
were generated with Rstudio 1.2.5001 and Inkscape (version 0.92, https://inkscape.org/). 403
404
Supplemental Material 405
Figure S1. The CherryTemp system can be effectively used to quickly alter intracellular dynamics in plant 406
cells. 407
Figure S2. Lowering the temperature increases the lifetime of endocytic proteins at the plasma membrane. 408
Figure S3. Lowering the temperature decreases the recruitment dynamics of the endocytic proteins. 409
Figure S4. Lowering the temperature enhances the temporal resolution of recruitment. 410
Figure S5. TWD40-1 localizes to the PM as endocytic foci. 411
Figure S6. TPLATE and TML are recruited to the PM simultaneously. 412
Figure S7. AtEHs and TPLATE are recruited to the PM simultaneously. 413
414
415
Acknowledgements 416
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Research in the Van Damme lab is funded by the European Research Council T-Rex project number 417
682436 and by the Chinese Scholarship Council (CSC) (scholarship 201508440249 to J.W.). A.J. is 418
supported by funding from the Austrian Science Fund (FWF): I3630B25 to J.F. 419
420
Declaration of Interests 421
The authors declare no competing interests. 422
423
Figure legends 424
Figure 1. Endocytic dynamics alter immediately upon lowering the temperature. 425
Spinning disc images and representative kymographs showing endocytic foci and lifetimes of CLC2 (left) or TPLATE (right) in 426
Arabidopsis etiolated hypocotyl epidermal cells at different temperatures. Cells were imaged at 25 °C for 2 minutes and then imaged 427
at 12 °C for an additional 3 minutes. Images were acquired with a 1-second interval. White arrows on the kymographs indicate the 428
position of the temperature shift. Scale bars of spinning disc images, 5 µm. Scale bars of kymographs, 30 µm. Time, 300s. 429
Figure 2. Lowering the temperature slows down CME. 430
(A) Spinning disk images and density quantification of TPLATE, TML and DRP1a-marked foci in epidermal hypocotyl cells at the 431
permissive (25 degrees) and restrictive (12 degrees) temperature. The density of the foci is independent of the tested temperatures 432
(n.s. not significant, Mann-Whitney U-test). The mean difference with the bootstrap 95% confidential interval (green circle and green 433
line) is shown as a part of the plot. Hedges’ g value is a standardized effect size. For each transgenic line, 8 cells from 8 seedlings 434
were imaged at both temperatures. Scale bars, 5 µm. (B) Representative kymographs and histograms showing the life-time 435
distribution of TPLATE, TML and DRP1a positive foci at both temperatures. Scale bars, 25 µm. Time, 120 s. Number of events (n) is 436
indicated. (C) Representative confocal images and Jitter and box plot quantification showing internalization of the FM4-64 dye (4μM, 437
30mins) in Col-0 root epidermal cells at both temperatures. The red circle represents the mean. The p-value was calculated by the 438
Welch two sample T-test. The mean difference with the bootstrap 95% confidential interval (green circle and green line) is shown on 439
the right side of the plot. Hedges’ g value is a standardized effect size. n represents the number of measurements (n = 43 cells for 440
25 °C and n = 48 cells for 12 °C) from 11 individual roots respectively. Scale bar, 10 µm. 441
Figure 3. Lowering the temperature enhances the temporal resolution of plasma membrane recruitment. 442
(A and D) Representative time series of dual-color spinning disk movies (2s/f) showing the sequential recruitment between TPLATE 443
and DRP1a or CLC2 at the different temperatures. Arrowheads mark the appearance of TPLATE or DRP1a and CLC2 at PM. (B and 444
E) Representative kymographs displaying the differential recruitment between TPLATE and DRP1a or CLC2 at different 445
temperatures. Scale bar, 25 µm. Time, 120s. (C and F) Paired comparison of the lifetimes of particular protein pairs at the different 446
temperatures. Each line represents an individual pair with the mean (white circle) ± SD (black line) on the sides of each plot for 447
different combinations of constructs and temperatures. The p-value was calculated using the Wilcoxon Signed Rank test. The green 448
circle in the bottom graph represents the paired mean difference with the bootstrap 95% confidential interval (green line). Hedges’ g 449
value is a standardized effect size. n represents the number of events. 450
Figure 4. Colocalization analysis hints at a tight association between core and peripheral TPC subunits at the PM. 451
Representative spinning disk dual-color images and corresponding pie charts displaying the percentage of colocalization of dual-452
labeled endocytic foci. TPLATE was compared to a late endocytic marker, DRP1a (A), to both TML (B) and TWD40-1 (C), and to 453
both AtEHs/Pan1 (D, E). Rotated TPLATE (90° rotation; TPLATE_R) was also compared to AtEH1/Pan1 to control for random 454
association of foci (F). Scale bars, 5 µm. Z-projections of five consecutive frames with average intensity were used for quantification. 455
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Quantification (%) of colocalization as calculated using the DIANA algorithm. The high percentage of overlap between TPLATE and 456
the AtEH/Pan1 proteins suggests a tight connection between those proteins at the plasma membrane. 457
Figure 5. The AtEHs/Pan1 and the core TPC subunits are simultaneously recruited to the PM 458
(A, D and G) Representative time series of dual-color spinning disk movies (2s/f) showing the recruitment between TPLATE and 459
TML, EH1/Pan1 and EH2/Pan1 at different temperatures. Arrows mark the appearance of TPLATE or TML, EH1/Pan1 and 460
EH2/Pan1 on the PM. (B, E and H) Representative kymographs displaying the recruitment of TPLATE versus TML, EH1/Pan1 and 461
EH2/Pan1 at different temperatures. Scale bar, 25 µm. Time, 120s. (C, F and I) Paired comparison of the life-times of particular 462
protein pairs at different temperatures. Each line on the sides of each plot represents an individual pair with the mean (white circle) ± 463
SD (black line) for different combinations of constructs and temperatures. The p-value was calculated by the Wilcoxon Signed Rank 464
test. The green circle in the bottom graph represents the paired mean difference with the bootstrap 95% confidential interval (green 465
line). Hedges’ g value is a standardized effect size. n represents the number of events. 466
Table 1. twd40-1-1 plants expressing C-terminal fusions of TWD40-1 with mRuby3 allow transfer of the T-DNA via the male. 467
The table shows the result of the analysis of back-cross experiments between Col-0 as female (♀ and the twd40-1-1 heterozygous 468
plants with or without expression of TWD40-1-mRuby3 as male (♂). The analysis clearly shows that the mutation blocks transfer via 469
the male and that this block is lifted by the presence of the fluorescent fusion construct, indicating that it is functional. 470
471
472
473
474
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Van Damme, D., Coutuer, S., De Rycke, R., Bouget, F.Y., Inze, D., and Geelen, D. (2006). Somatic 590 cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat 591 proteins. Plant Cell 18, 3502-3518. 592 Van Damme, D., Gadeyne, A., Vanstraelen, M., Inze, D., Van Montagu, M.C., De Jaeger, G., Russinova, E., 593 and Geelen, D. (2011). Adaptin-like protein TPLATE and clathrin recruitment during plant somatic 594 cytokinesis occurs via two distinct pathways. Proc Natl Acad Sci U S A 108, 615-620. 595 Wang, P., Pleskot, R., Zang, J., Winkler, J., Wang, J., Yperman, K., Zhang, T., Wang, K., Gong, J., Guan, Y., 596 et al. (2019). Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin 597 and endocytic machinery. Nat Commun 10, 5132. 598 Wrobel, A.G., Kadlecova, Z., Kamenicky, J., Yang, J.C., Herrmann, T., Kelly, B.T., McCoy, A.J., Evans, P.R., 599 Martin, S., Muller, S., et al. (2019). Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via 600 AP2 Phosphorylation. Dev Cell 50, 494-508 e411. 601 Yamaoka, S., Shimono, Y., Shirakawa, M., Fukao, Y., Kawase, T., Hatsugai, N., Tamura, K., Shimada, T., and 602 Hara-Nishimura, I. (2013). Identification and dynamics of Arabidopsis adaptor protein-2 complex and its 603 involvement in floral organ development. Plant Cell 25, 2958-2969. 604 Zhang, Y., Persson, S., Hirst, J., Robinson, M.S., van Damme, D., and Sanchez-Rodriguez, C. (2015). 605 Change your TPLATE, change your fate: plant CME and beyond. Trends Plant Sci 20, 41-48. 606 Zhou, J., Liu, D., Wang, P., Ma, X., Lin, W., Chen, S., Mishev, K., Lu, D., Kumar, R., Vanhoutte, I., et al. 607 (2018). Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-608 box PUB12/PUB13-mediated ubiquitination. Proc Natl Acad Sci U S A 115, E1906-E1915. 609
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12 °C
25 °CC
LC
-mK
Osingle frame distance (µm)
25 °C
12 °C 12 °C
TP
LA
TE
-GF
P
single frame
25 °C
distance (µm)
tim
e (s
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12 °Ctim
e (s
)
Figure 1. Endocytic dynamics alter immediately upon lowering the temperature.Spinning disc images and representative kymographs showing endocytic foci and lifetimes of CLC2 (left) or TPLATE (right) in Arabidopsis etiolated hypocotyl epidermal cells at different temperatures. Cells were imaged at 25 °C for 2 minutes and then imaged at 12 °C for an additional 3 minutes. Images were acquired with a 1-second interval. White arrows on the kymographs indicate the position of the temperature shift. Scale bars of spinning disc images, 5 µm. Scale bars of kymographs, 30 µm. Time, 300s.
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Figure 2. Lowering the temperature slows down CME.(A) Spinning disk images and density quantification of TPLATE, TML and DRP1a-marked foci in epidermal hypocotyl cells at the permissive (25 degrees) and restrictive (12 degrees) temperature. The density of the foci is independent of the tested temperatures (n.s. not significant, Mann-Whitney U-test). The mean difference with the bootstrap 95% confidential interval (green circle and green line) is shown as a part of the plot. Hedges’ g value is a standardized effect size. For each transgenic line, 8 cells from 8 seedlings were imaged at both temperatures. Scale bars, 5 µm. (B) Representative kymographs and histograms showing the life-time distribution of TPLATE, TML and DRP1a positive foci at both temperatures. Scale bars, 25 µm. Time, 120 s. Number of events (n) is indicated. (C) Representative confocal images and Jitter and box plot quantification showing internalization of the FM4-64 dye (4μM, 30mins) in Col-0 root epidermal cells at both temperatures. The red circle represents the mean. The p-value was calculated by the Welch two sample T-test. The mean difference with the bootstrap 95% confidential interval (green circle and green line) is shown on the right side of the plot. Hedges’ g value is a standardized effect size. n represents the number of measurements (n = 43 cells for 25 °C and n = 48 cells for 12 °C) from 11 individual roots respectively. Scale bar, 10 µm.
A
25 °
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TPLATE TML DRP1a
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ten
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lati
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o P
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n = 43 n = 48
p = 0.00355Hedges' g - 0.6190 (medium)
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n d
iffe
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10
-10p = 0.958Hedges' g - 0.0128
(negligible)
p = 0.227Hedges' g 0.384
(small)
p = 0.318Hedges' g - 0.31
(small)
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Merge
TPLATE
CLC2
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CLC2
Lif
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Mea
n d
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TPLATE DRP1aTPLATE DRP1a
9
10
1129
30
31 p-value < 2.2e-16
Hedge's g - 1.013
(large)
n = 660
p-value < 2.2e-16
Hedge's g - 1.271
(large)
n = 743
0
1
2DRP1a - TPLATE
25 °C
DRP1a - TPLATE
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Merge
DRP1a
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C
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DRP1a
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Merge
DRP1a
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B distance (µm)
tim
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me
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e (s
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distance (µm)
tim
e (s
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A C
ED F
p-value = 9.3e-10
Hedge's g - 0.170
(small)
n = 157
p-value < 2.2e-16
Hedge's g - 0.143
(small)
n = 470
27s - 47s
1s - 21s
3s - 23s
Figure 3. Lowering the temperature enhances the temporal resolution of plasma membrane recruitment. (A and D) Representative time series of dual-color spinning disk movies (2s/f) showing the sequential recruitment between TPLATE and DRP1a or CLC2 at the different temperatures. Arrowheads mark the appearance of TPLATE or DRP1a and CLC2 at PM. (B and E) Representative kymographs displaying the differential recruitment between TPLATE and DRP1a or CLC2 at different temperatures. Scale bar, 25 µm. Time, 120s. (C and F) Paired comparison of the lifetimes of particular protein pairs at the different temperatures. Each line represents an individual pair with the mean (white circle) ± SD (black line) on the sides of each plot for different combinations of constructs and temperatures. The p-value was calculated using the Wilcoxon Signed Rank test. The green circle in the bottom graph represents the paired mean difference with the bootstrap 95% confidential interval (green line). Hedges’ g value is a standardized effect size. n represents the number of events.
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A
B
TPLATE DRP1a Merge% Colocalization
% Only DRP1a
% Only TPLATE
n = 2075
40.8% 56.6%
2.6%
TML TPLATE Merge
18.9%
77.2%
3.9%
% Colocalization
% Only TPLATE
% Only TML
n = 2180
TPLATE EH1 Merge
90.2%
7.1% 2.7%
% Colocalization
% Only EH1
% Only TPLATE
n = 2405
TPLATE WD40-1 Merge
9.3% 9.6%
81.1% % Colocalization
% Only WD40-1
% Only TPLATE
n = 5907
C
D
E TPLATE EH2 Merge
6.3%
% Colocalization
% Only EH2
% Only TPLATE
n = 23246.9%
86.8%
F% Colocalization
% Only EH1
% Only TPLATE_R
n = 2165
TPLATE_R EH1 Merge
11%
40%49%
Figure 4. Colocalization analysis hints at a tight association between core and peripheral TPC subunits at the PM. Representative spinning disk dual-color images and corresponding pie charts displaying the percentage of colocalization of dual-labeled endocytic foci. TPLATE was compared to a late endocytic marker, DRP1a (A), to both TML (B) and TWD40-1 (C), and to both AtEHs/Pan1 (D, E). Rotated TPLATE (90° rotation; TPLATE_R) was also compared to AtEH1/Pan1 to control for random association of foci (F). Scale bars, 5 µm. Z-projections of five consecutive frames with average intensity were used for quantification. Quantification (%) of colocalization as calculated using the DIANA algorithm. The high percentage of overlap between TPLATE and the AtEH/Pan1 proteins suggests a tight connection between those proteins at the plasma membrane.
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distance (µm)
tim
e (s
)
E
*
25 º
C
Merge
TPLATE
EH1
distance (µm)
tim
e (s
)
D
Lif
etim
e (s
)
75
50
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TPLATE EH1
25 °C 12 °C
100
125
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175
Mea
n d
iffe
ren
ce (
s) 0
1.0
2.0
3.0
4.0
5.0
EH1 - TPLATE25 °C
EH1 - TPLATE12 °C
p = 0.00035Hedge's g - 0.023
(negligible)n = 309
p = 2.1e-08Hedge's g - 0.034
(negligible)n = 383
200
TPLATE EH1
F
12 º
C
Merge
TPLATE
EH1
distance (µm)
tim
e (s
)
25 º
C
Merge
TML
TPLATE
A
distance (µm)
tim
e (s
)
distance (µm)
tim
e (s
)
Lif
etim
e (s
)
75
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TML TPLATE
25 °C 12 °C
100
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175
Mea
n d
iffe
ren
ce (
s) 0
1.0
2.0
3.0
4.0
5.0
TPLATE - TML25 °C
TPLATE - TML12 °C
p = 0.5869Hedge's g - 0.015
(negligible)n = 376
p = 0.001118Hedge's g - 0.055
(negligible)n = 317
TML TPLATE
C200
12 º
C TML
TPLATE
Merge
B
Lif
etim
e (s
)
75
50
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TPLATE EH2
25 °C 12 °C
100
125
150
175
Mea
n d
iffe
ren
ce (
s) 0
1.0
2.0
3.0
4.0
5.0
EH2 - TPLATE25 °C
EH2 - TPLATE12 °C
p = 0.022Hedge's g - 0.019
(negligible)n = 308
p = 6.00e-06Hedge's g - 0.030
(negligible)n = 317
200
TPLATE EH2
Idistance (µm)
tim
e (s
)
Merge
TPLATE
EH2
25 º
C
HG
Merge
TPLATE
EH2
12 º
C
TML - TPLATE_25 ºC
TPLATE - EH1_25 ºC
TPLATE - EH1_12 ºC
TPLATE - EH2_25 ºC
TPLATE - EH2_12 ºC
TML - TPLATE_12 ºC
7s - 15s
1s - 9s
7s - 15s
3s - 11s
Figure 5. The AtEHs/Pan1 and the core TPC subunits are simultaneously recruited to the PM(A, D and G) Representative time series of dual-color spinning disk movies (2s/f) showing the recruitment between TPLATE and TML, EH1/Pan1 and EH2/Pan1 at different temperatures. Arrows mark the appearance of TPLATE or TML, EH1/Pan1 and EH2/Pan1 on the PM. (B, E and H) Representative kymographs displaying the recruitment of TPLATE versus TML, EH1/Pan1 and EH2/Pan1 at different temperatures. Scale bar, 25 µm. Time, 120s. (C, F and I) Paired comparison of the life-times of particular protein pairs at different temperatures. Each line on the sides of each plot represents an individual pair with the mean (white circle) ± SD (black line) for different combinations of constructs and temperatures. The p-value was calculated by the Wilcoxon Signed Rank test. The green circle in the bottom graph represents the paired mean difference with the bootstrap 95% confidential interval (green line). Hedges’ g value is a standardized effect size. n represents the number of events.
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