Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features
- University of California, Berkeley, United States
- Yale University, United States
Figures
Figure 1 with 3 supplements
Quantitative side-by-side comparison of levels of homologous proteins at endocytic sites in budding and fission yeast.
Single frames from unprocessed (left panel) and processed (middle panel) movies for particle intensity quantification, and average maximum intensity of indicated proteins at endocytic sites (right …
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Figure 1—source data 1
Figure 1A-G maximum intensity source data.
- https://cdn.elifesciences.org/articles/50749/elife-50749-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Endocytic sites in budding and fission yeast: A brief summary of what has been reported previously.
The endocytic actin machinery on invaginated membranes in budding (A) or fission (B) yeast. (A) In budding yeast, actin filaments are proposed to be nucleated near the base of the invagination. …
Figure 1—figure supplement 2
Quantitative comparison of fluorescently-tagged scSla1 in budding yeast cultured in different media.
(A) Budding yeast expressing Sla1-GFP were cultured in budding yeast (upper panel) or fission yeast (lower panel) media. The cells were then imaged by the Materials and methods commonly used for …
Figure 1—figure supplement 3
Live cell imaging data processing and analysis by Particle Tracker plugin.
(A) Single frame from movie processed for background subtraction and photobleaching correction (left panel). A median filter was used to compute the local background surrounding the endocytic …
Figure 2 with 3 supplements
Determining the maximum number of fluorescently-tagged endocytic proteins at endocytic sties by ratiometric comparison of fluorescence intensities to the intensity of 120-sfGFP-tagged nanocages.
(A) Comparison of ratios of maximum protein levels at endocytic sites for the two yeasts in this study (green) vs in previous studies (gray). (B) The mean fluorescence intensity of 120-sfGFP- tagged …
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Figure 2—source data 1
Figure 2D maximum protein number source data.
- https://cdn.elifesciences.org/articles/50749/elife-50749-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Quantitative comparison of 120-sfGFP-tagged nanocages prepared on different days.
120-sfGFP-tagged nanocages were prepared and imaged three different days using the same method and conditions. The fluorescence intensities of the 120-sfGFP-tagged nanocages were measured and …
Figure 2—figure supplement 2
Quantitative comparison of the maximum number of homologous proteins at endocytic sites in budding and fission yeast.
(A-I) Cells expressing indicated proteins were mixed and then imaged over time. The resulting movies were analyzed using a particle tracking program. The maximum fluorescence intensities were …
Figure 2—figure supplement 3
Determining the maximum number of Myo3-GFP molecules using Sla1-GFP and Las17-GFP as standards.
(A) Determining the maximum number of Myo3-GFP molecules using Las17-GFP as a standard. Two-color image of MYO3-GFP ABP1-RFP cells and LAS17-GFP cells (left panel). Single frame of a movie of MYO3-GF…
Figure 3 with 1 supplement
Quantitative comparison of endocytic actin dynamics in budding and fission yeast.
(A) Single frame from a movie of simultaneously imaged scABP1-GFP in budding yeast and spfim1-mEGFP in fission yeast. (B and C) Single endocytic events represented by scAbp1 in budding yeast (B) and …
Figure 3—figure supplement 1
Predicting the timing of endocytic vesicle scission by the changes of the standard deviation of actin patch displacement.
(A) The standard deviation (SD) of actin patch displacement starts to greatly increase when scAbp1 patches move ~125 nm away from their origin. The light blue line represents the average …
Figure 4
Quantitative comparison of endocytic coat dynamics for budding and fission yeast.
(A and B) Single endocytic event detected by tracking fluorescent scSla1 in budding yeast (A) or spPan1 in fission yeast (B). Graphs from left to right: protein patch centroid position over time, …
Figure 5 with 2 supplements
Spatial-temporal relationship of coat proteins, scission proteins and the actin network at fission yeast endocytic sites.
(A) Single frame from a movie of spPan1-mEGFP and spFim1-mCherry expressed in fission yeast cells. (B) Alignment of average intensity (solid lines) and displacement (dotted lines) of spPan1-mEGFP …
Figure 5—figure supplement 1
Alignment and quantification of average trajectories for spFim1-mCherry and spPan1-mEGFP in fission yeast.
(A) Alignment of intensity and displacement of spPan1-mEGFP and spFim1-mCherry for a single endocytic event. Two examples are presented. (B) Alignment of average intensity and displacement plots for …
Figure 5—figure supplement 2
Alignment and quantification of average trajectories for spFim1-mCherry and spHob1-GFP in fission yeast.
(A) Alignment of intensity and displacement of spHob1-GFP and spFim1-mCherry for a single endocytic event. Two examples are presented. (B) Alignment of average intensity and displacement plots for …
Figure 6 with 1 supplement
3D-STORM imaging of coat protein and the actin network at fission yeast endocytic sites.
(A and B) The length of nanoscale structures of spFim1 (n = 92) or spPan1 (n = 46) at endocytic sites revealed by STORM analysis was measured and plotted. Representative STORM images of different …
Figure 6—figure supplement 1
3D-STORM imaging of spFim1 and spPan1.
(A) Chemically fixed fission yeast cells expressing mEGFP-tagged proteins were labeled with AF647-conjugated anti-GFP nanobodies and imaged at the equatorial plane. (B) Conventional and 3D-STORM …
Figure 7 with 3 supplements
Spatio-temporal relationship between two major NPFs and endocytic actin assembly in fission yeast.
(A) Dynamics of spWsp1. Numerous endocytic events represented by mEGFP-spWsp1 were analyzed and aligned. Graphs from left to right: (right) Protein position vs time for endocytic events (n = 31) …
Figure 7—figure supplement 1
Dynamics of GFP-scLas17 and scLas17-GFP in budding yeast.
(A) Dynamics of scLas17-GFP and GFP-scLas17. Single frames (left) from movies, radial kymograph representations (Sun et al., 2017) (middle), and time series showing progression of a single endocytic …
Figure 7—figure supplement 2
Alignment and quantification for average trajectories for spFim1-mCherry and mEGFP-spWsp1 in fission yeast.
(A) Alignment of intensity and displacement for mEGFP-spWsp1 and spFim1-mCherry for a single endocytic event. Two examples are presented. (B) Alignment of average intensity and displacement of …
Figure 7—figure supplement 3
Alignment and quantification for average trajectories for spFim1-mCherry and mEGFP-spMyo1- in fission yeast.
(A) Alignment of intensity and displacement for mEGFP-spMyo1 and spFim1-mCherry for a single endocytic event. Two examples are presented. (B) Alignment of average intensity and displacement for …
Figure 8
Functional analysis of CA domains in two fission yeast nucleation promoting factors.
(A) Dynamics of spWsp1CA∆-GFP patches. Single frame from movie of cells expressing mEGFP-spWsp1 (left upper panel) or spWsp1-CA∆-GFP (left bottom panel). Alignment of average intensity and …
Figure 9
Comparison of endocytic vesicle formation in fission and budding yeast.
Timeline and summary of the average molecule numbers for indicated coat proteins and actin machinery components in fission (A) and budding yeast (B) endocytosis. Scission occurs at the time 0. Steps …
Author response image 1
Author response image 2
Videos
Video 1
Dynamics of mEGFP-spWsp1 and spFim1-mCherry in mEGFP-spwsp1 spfim1-mCherry S. pombe cells.
Time to acquire one image pair was 136 ms. Interval between frames is 548 ms.
Video 2
Dynamics of spwsp1CA∆-GFP and spFim1-mCherry in spwsp1-CA∆-GFP spfim1-mCherry S. pombe cells.
Time to acquire one image pair was 400 ms. Interval between frames is 460 ms.
Video 3
Dynamics of mEGFP-spMyo1 and spFim1-mCherry in mEGFP-myo1 spfim1-mCherry S. pombe cells.
Time to acquire one image pair was 200 ms. Interval between frames is 222 ms.
Video 4
Dynamics of spMyo1CA∆-GFP and spFim1-mCherry in spmyo1CA∆-GFP spfim1-mCherry S. pombe cells.
Time to acquire one image pair was 200 ms. Interval between frames is 222 ms.
Tables
Table 1
Comparison of peak protein numbers in actin patches.
Calibration methods: Sirotkin, Arasada, Chen, Epstein, MacQuarrie: a range of internal standards tagged with YFP or GFP; Basu: comparison with actin patches in other cells marked with Arp2/3 complex …
| Protein | S. pombe | S. cerevisiae | ||||||
|---|---|---|---|---|---|---|---|---|
| Sirotkin et al. (2010)a; Arasada and Pollard (2011)b; Chen and Pollard (2013)c; Epstein et al. (2018)d | Basu and Chang (2011) | MacQuarrie et al. (2019) | Sun This study | Sun This study | Picco et al. (2015) Rvs167 125 | Manenschijn et al. (2019) | Galletta et al. (2012) | |
| Actin | 7500 a; 4050 b | 3563 | ||||||
| Arp2/3 complex subunits | 320, 320, 320 a | ~150 | 304 (302) | 294 | 210 | 247 | 300 | |
| WASp/Las17 | 230 a; 140 b; 135 c | 125 | ~150 | 138 | 102 | 46 | ||
| WIP/Vrp1 | 140 a | 95 | 78 | |||||
| Myosin-I/ Myo3, Myo5 | 400 a; 170 b; 240 c | ~350 | 170 | Myo5: 200 Myo3: 100 | Myo5: 132 | Myo5: ~130 Myo3: ~60 | ||
| Fimbrin/Sac6 | 910 a; 800 c; 1000, 1600 d | 675 | 545 | 455 | ||||
| End4/Sla2 (HIP1R) | 160 a; 70 c | 124 | 133 | 37, 47 | ~40 | |||
| Pan1/End3 (Intersectin) | Pan1: 260a; 160 c | Pan1: 219 | Pan1: 131 End3: 100 | End3: 60 | ||||
| Sla1 | 168 | 91 | ~90 | |||||
| ABP1 | 800 | 423 | 515 | 810 | ||||
| Clathrin HC | 40 a | |||||||
| Clathrin LC | 40, 30 a | ~40 | ||||||
| Capping protein | 230 a | 197 | 200 | |||||
| App1 | 150 a | |||||||
| Coronin | 490 a; 320 b | |||||||
| Twinfillin | 210 a | |||||||
| F-BAR Cdc15 | 125 b | ~70 | ||||||
| F-BAR Bzz1 | 90 b | |||||||
| Dip1 | 20 | |||||||
| Bbc1 | ~50 | |||||||
| Amphiphysin | Rvs167 125 | |||||||
Table 2
The ratio of peak fluorescence intensities between various fluorescently tagged proteins.
| GFP-tagged protein pair | The ratio of peak fluorescence intensities |
|---|---|
| spPan1: scSla1 | 1.30: 1 |
| spPan1: scPan1 | 1.67: 1 |
| scEnd3: spPan1 | 0.46: 1 |
| scSla2: scLas17 | 1.30: 1 |
| spEnd4: scSla2 | 0.93: 1 |
| spWsp: scLas17 | 1.40: 1 |
| scVrp1: scSla1 | 0.47: 1 |
| spVrp1: scVrp1 | 1.23: 1 |
| scMyo3: scMyo5 | 0.51: 1 |
| spMyo1: scMyo3 and scMyo5 | 0.57: 1 |
| spArc5: scSla1 | 1.80: 1 |
| spArc5: scArc15 | 1.03: 1 |
| spArc3: scArc15 | 1.03: 1 |
| scAbp1: scSla1 | 4.80: 1 |
| spFim1: scAbp1 | 0.84: 1 |
| spFim1: scSac6 | 1.24: 1 |
Table 3
Peak molecules per patch vs total molecules per cell for various endocytic proteins in budding and fission yeast.
| A. Peak molecules per patch vs total molecules per cell in budding yeast | |||
|---|---|---|---|
| S. cerevisiae Protein | Total molecules per cell | Peak molecules per patch | Ratio |
| Ho et al. (2018) | Sun (This study) | ||
| Arp2/3 complex subunits | 12,784, | 294 | 0.023 |
| WASp/Las17 | 3134 | 102 | 0.033 |
| WIP/Vrp1 | 3559 | 78 | 0.021 |
| Myosin-I/ Myo3 and Myo5 | Myo5, 6051 Myo3, 4222 | Myo5:200 Myo3:100 | 0.033 0.024 |
| Fimbrin/Sac6 | 24,543 | 545 | 0.022 |
| HIP1R/Sla2 | 8879 | 133 | 0.015 |
| Intersectin /Pan1 and End3 | Pan1:9830 End3:6064 | Pan1: 131 End3:100 | 0.013 0.016 |
| Sla1 | 8776 | 91 | 0.010 |
| Abp1 | 18,301 | 800 | 0.044 |
| B. Peak molecules per patch vs total molecules per cell in fission yeast | |||
| S. pombe Protein | Total molecules per cell | Peak molecules per patch | Ratio |
| Sirotkin et al., 2010; Wu and Pollard, 2005 | Sun (This study) | ||
| Arp2/3 complex subunits | ~40,000 | 304 (302) | 0.008 |
| WASp/Wsp1 | 68,000 | 138 | 0.002 |
| WIP/Vrp1 | 19,000 | 95 | 0.005 |
| Myosin-I | 63,000 | 170 | 0.003 |
| Fimbrin/Fim1 | 87,000 | 675 | 0.008 |
| HIP1R/End4 | 22,000 | 124 | 0.006 |
| Intersectin/Pan1 | 27,000 | 219 | 0.008 |
Table 4
Strains used in this study.
| DDY2733* | MATa his3-∆200 leu2-3, 112 lys2-801 ura3-52 ABP1-GFP::HIS3 |
| DDY2734* | MATa his3-∆200 leu2-3, 112 lys2-801 ura3-52 SLA1-GFP::HIS3 |
| DDY2735* | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 PAN1-GFP::HIS3 |
| DDY2795* | MATa his3-∆200 leu2-3, 112 lys2-801 ura3-52 SLA2-GFP::HIS3 |
| DDY2736* | MATa his3-∆200 leu2-3, 112 lys2-801 ura3-52 LAS17-GFP::HIS3 |
| DDY3201* | MATa his3-∆200 leu2-3, 112 lys2-801 ura3-52 VRP1-GFP::HIS3 |
| DDY2752* | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 ARC15-GFP::HIS3 |
| DDY2960* | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 SAC6-GFP::HIS3 |
| YSY4303 | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 LAS17-sfGFP::HIS3 |
| YSY4305 | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 MYO5-sfGFP::HIS3 |
| YSY4307 | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 SLA1-sfGFP::HIS3 |
| YSY4315 | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 CSE4-sfGFP::HIS3 |
| YSY4218 | MATα his3-∆200 leu2-3, 112 lys2-801 ura3-52 MYO3-GFP::HIS3 |
| YSY4299 | MAT a his3-∆200 leu2-3, 112 lys2-801 ura3-52 MYO3-GFP::HIS, ABP1-RFP::HygMX |
| YSY4300 | MAT α his3-∆200 leu2-3, 112 lys2-801 ura3-52 MYO5-GFP::HIS, ABP1-RFP::HygMX |
| YSY4125 | MAT a his3-∆200 leu2-3, 112 lys2-801 ura3-52 GFP-LAS17::KanMX6, ABP1-RFP::HygMX |
| YSY4126 | MAT α his3-∆200 leu2-3, 112 lys2-801 ura3-52 GFP-LAS17::KanMX6, MYO5-RFP::HIS |
| YSY4291 | MAT α his3-∆200 leu2-3, 112 lys2-801 ura3-52 Myo5-GFP::KanMX6, MYO3-GFP::HIS |
| YSY4120 | MAT α his3-∆200 leu2-3, 112 lys2-801 ura3-52 GFP-LAS17::KanMX6 |
| YSY4257 | h+ leu1-32 ura4-D18 his3-D1 ade6-M210 pan1-mEGFP-kanMX6 fim1mcherry-natRMX6 |
| YSY4266 | h- or h+ leu1-32 ura4-D18 his3-D1 ade6 kanMX6- Pwsp1-mEGFP-wsp1 fim1mcherry-natRMX6 |
| YSY4269 | h- or h+ leu1-32 ura4-D18 his3-D1 ade6 kanMX6- Pmyo1-mEGFP-myo1 fim1mcherry-natRMX6 |
| YSY4335 | h+ leu1-32 ura4-D18 his3-D1 ade6-M210 fim1mcherry-natRMX6 wsp1-CA∆-GFP-kanMX6 |
| YSY4345 | h- or h+ leu1-32 ura4-D18 his3-D1 ade6-M210 myo1-ca∆-GFP-kanMX6 fim1-mcherry-natRMX6 |
| YSY4346 | h- leu1-32 ura4-D18 his3-D1 ade6-M210 fim1mcherry-natRMX6, hob1-GFP-kanMX6 |
| YSY4346 | h+ leu1-32 ura4-D18 his3-D1 ade6-M210 hob1-GFP-kanMX6 |
| TP401** | h+ leu1-32 ura4-D18 his3-D1 ade6-M210 pan1-mEGFP-kanMX6 |
| TP398** | h+ leu1-32 ura4-D18 his3-D1 ade6-M210 end4-mEGFP-kanMX6 |
| TP203** | h- leu1-32 ura4-D18 his3-D1 ade6-M210 kanMX6- Pwsp1-mEGFP-wsp1 |
| TP186** | h- or h+ leu1-32 ura4-D18 his3-D1 ade6-M210 vrp1-EGFP-kanMX6 |
| TP198** | h- leu1-32 ura4-D18 his3-D1 ade6-M210 kanMX6-myo1-mEGFP |
| TP226** | h- leu1-32 ura4-D18 his3-D1 ade6-M210 arpc5-mEGFP-kanMX6 |
| TP347** | h- leu1-32 ura4-D18 ade6-M210 fim1-mEGFP-kanMX |
| * | from Kaksonen et al. (2003) or Sun et al. (2006) |
| ** | from Sirotkin et al. (2005) or Wu and Pollard (2005) |
| otherwise | This study |
