EMBO/soda-nel huggingface dataset:
Precise Control of Microtubule Disassembly in Living Cells (2022)
Figure 1

Figure 1. Recruitment of engineered Spastins onto microtubules leads to their rapid disassembly

Schematic of the inducible microtubule disassembly system. Dimerization of three different pairs FRB/FKBP, GAIs/mGID1, and Cry2/CIBN can be triggered by rapamycin (Rapa), GA3-AM, and blue light, respectively. One of the dimerizing domains (DDs) is fused with one of two engineered Spastin enzymes, dNSpastin3Q and C2Lact-tagged dNSpastin, whereas the other is fused with microtubule-associated proteins (MAPs). Dimerization upon certain stimuli (e.g., chemical treatments or light illumination) induces recruitment of engineered Spastins from cytosol or plasma membrane (PM) onto MAP-labeled microtubules. Acute accumulation of Spastins on microtubules rapidly induces disassembly of target microtubules.

HeLa cells co-transfected with EMTB-CFP-FRB and YFP-FKBP were treated with 100 nM rapamycin (Rapa). Addition of rapamycin rapidly translocated YFP-FKBP onto EMTB-CFP-FRB-labeled microtubules and increased the FRET signal. The scale for the FRET/CFP intensity ratio is shown. Scale bar, 10 μm.

The normalized intensity of FRET/CFP in cells before and after rapamycin (blue) and 0.1% DMSO (control; red) treatment. n = 6 and 10 cells in the rapamycin and DMSO groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.

The video frames of microtubule disruption upon the recruitment of dNSpastin3Q-YFP-FKBP onto microtubules. HeLa cells co-transfected with the indicated constructs were treated with rapamycin (100 nM). Scale bar, 10 μm.

The normalized microtubule filament area in cells with different microtubule disruption treatments. n = 30, 45, 19, 20, 14, and 6 cells in nocodazole (3.3 µM), colchicine (500 µM), dNSpastin-YFP-FKBP-C2Lact, dNSpastin3Q-YFP-FKBP, dNSpastin3QED-YFP-FKBP, and dNSpastin3Q-YFP-FKBP with MG132 (50 μM) pre-treatment, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.

HeLa cells co-transfected with the indicated constructs were treated with nocodazole (Noc, 3.3 µM; left) or rapamycin (Rapa, 100 nM; right) for 0, 30, and 60 min. Transfected cells were then fixed and labeled with anti-acetylated tubulin (red). Dotted lines indicate the boundary of transfected cells. Scale bars, 10 μm.

The intensity of acetylated tubulin in cells transfected with EMTB-CFP-FRB (orange) or cotransfected with EMTB-CFP-FRB and dNSpastin3Q-YFP-FKBP (blue) after addition of nocodazole (Noc, 3.3 µM) or rapamycin (Rapa, 100 nM) for the indicated times. n = 123 and 81 cells in the nocodazole and dNSpastin3Q groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed, with p-values indicated.
Precise Control of Microtubule Disassembly in Living Cells (2022)
Figure 2

Figure 2. Rapid disruption of primary cilia, mitotic spindles, and intercellular bridges

NIH3T3 fibroblasts co-transfected with 5HT6-mCherry (5HT6-mCh; a ciliary membrane marker; red), CFP-FRB-MAP4m (blue), and dNSpastin3Q-YFP-FKBP (green) were serum-starved for 24 hr to induce ciliogenesis. Ciliated cells were then treated with rapamycin (Rapa, 100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to axonemal microtubules. Dotted lines indicate the cell boundaries. Scale bar, 5 μm.

The normalized length of axonemes (blue), primary cilia (red), and dNSpastin3Q-YFP-FKBP in cilia (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.

HeLa cells co-transfected with H2B-mCherry (H2B-mCh; a chromosome marker; red), CFP-FRB-MAP4m (a marker of mitotic spindles; blue), and dNSpastin3Q-YFP-FKBP (green) were synchronized in metaphase and treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to mitotic spindles. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.

The normalized area of the mitotic spindle (blue) and intensity of dNSpastin3Q-YFP-FKBP in the mitotic spindle (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.

HeLa cells co-transfected with H2B-mCherry (red), CFP-FRB-MAP4m (intercellular bridges; blue), and dNSpastin3Q-YFP-FKBP (green) were treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to intercellular bridges. Arrows indicate the intercellular bridges. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.

The normalized area of intercellular bridges (blue) and intensity of dNSpastin3Q-YFP-FKBP at intercellular bridges (green) upon rapamycin treatment. n = 5 cells from three independent experiments. Data are shown as the mean ± S.E.M.
Precise Control of Microtubule Disassembly in Living Cells (2022)
Figure 3

Figure 3. Disruption of tyrosinated microtubules

COS7 cells transfected with TagRFP-FRB-A1AY1 (red) were labeled by anti-α-tubulin antibody (green; upper panel), anti-tyrosinated tubulin antibody (green; middle panel), or anti-detyrosinated tubulin antibody (green; lower panel), respectively. Scale bar, 10 µm.

The intensity profiles of TagRFP-FRB-A1AY1 (red) and tubulin with the indicated PTMs (green) along dotted lines drawn in (A).

The Pearson's correlation coefficients for TagRFP-FRB-A1AY1 and indicated tubulin (tub) subtypes were calculated, and data are shown as the mean ± S.E.M. n = 16, 13, and 15 cells from left to right, respectively; three independent experiments.

COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and TagCFP-FKBP (green) were treated with rapamycin (100 nM). The addition of rapamycin rapidly recruits TagCFP-FKBP from cytosol onto A1AY1-labeled microtubules (arrows). Scale bar, 10 µm.

The normalized intensity of TagCFP-FKBP at A1AY1-labeled microtubules upon rapamycin treatments. n=7 cells from three independent experiments. Data are shown as the mean ± S.E.M.

COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and dNSpastin3Q-TagCFP-FKBP (cyan) were treated with 0.1% DMSO or rapamycin (100 nM) for 1 hr and followed by immunostaining with anti-tyrosinated tubulin antibody (green). Dotted lines highlight the transfected cells. Scale bar, 20 μm.

The normalized intensity of tyrosinated microtubules in TagRFP-FRB-A1AY1 and dNSpastin3Q-TagCFP-FKBP co-transfected cells upon 0.1% DMSO or rapamycin treatment for 1 hr. n=31 and 26 cells in DMSO and rapamycin treated groups, respectively, from three independent experiments. Data (blue) represent as mean ± S.E.M. Student's t-tests were performed with p values indicated.
Precise Control of Microtubule Disassembly in Living Cells (2022)
Figure 4

Figure 4. Using light to disassemble microtubules in a reversible and location-specific manner

COS7 cells co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red) were incubated with SPY650-tubulin (SPY650-tub) to visualize microtubules. The cells were illuminated by blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm.

The normalized intensity of mCh-Cry2 at microtubules in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.

COS7 cells co-transfected with EMTB-YFP-CIBN and dNSpastin3Q-mCh-Cry2 were incubated with SPY650-tubulin to visualize microtubules. The cells were illuminated by blue light as in (A). Scale bar, 10 μm.

The normalized intensity of dNSpastin3Q-mCh-Cry2 and mCh-Cry2 at microtubules and the normalized area of SPY650-tubulin in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastin3Q-mCh-Cry2 and mCh-Cry2 groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.
Precise Control of Microtubule Disassembly in Living Cells (2022)
Figure 5

Figure 5. The effects of acute microtubule disassembly on the dynamics and activities of mitochondria

COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Tom20-Neon (yellow), were treated with rapamycin for acute microtubule disruption. The dynamics of mitochondria at different levels of microtubule disruption are shown. Insets show higher-magnification images of mitochondrial fusion (a, filled arrows), fission (b, filled arrowheads), and unsuccessful fusion (c, d; hollow arrowheads) events as indicated by the dotted squares. Scale bar, 10 µm.

Single-cell analysis of local mitochondrial movement before and after microtubule disruption. n = 6119 mitochondria from nine cells, from four independent experiments. Data are shown as the mean ± S.E.M.

The frequency of mitochondrial fusion and fission events at different levels of microtubule disruption is shown. Data are shown as the mean ± S.E.M. n = 8 cells from three independent experiments.

The ratio of mitochondrial fusion and fission events at different levels of microtubule disruption is shown. Data are shown as the mean ± S.E.M. n = 8 cells from three independent experiments.

E-F. COS7 cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon were treated with rapamycin (100 nM) for acute microtubule disruption. The morphology of mitochondria at different levels of microtubule disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M.

G-H. The normalized intensity of MitoTracker Red in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB and dNSpastin3Q-YFP-FKBP or dNSpastin3QED-YFP-FKBP after rapamycin (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M.

Data information: Student's t-tests were performed with p values indicated.
Precise Control of Microtubule Disassembly in Living Cells (2022)
Figure 6

Figure 6. Microtubule disassembly enhances stress fiber formation and cell rigidity

COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Lifeact-Neon (heatmap and green) were treated with rapamycin (Rapa, 100 nM) to induce microtubule disassembly. Arrows and arrowheads indicate lamellipodia and stress fibers, respectively. Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals. Scale bar, 10 µm.

The normalized level of microtubule area, lamellipodia length, and filopodia density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.

Schematic representation of the hybrid atomic force microscope and confocal microscope system. A bright-field image of the atomic force microscopy (AFM) probe and an examined cell is shown. Dotted lines indicate the cell boundary. Scale bar, 10 µm.

Serial rigidity measurements of COS-7 cells that co-expressed EMTB-CFP-FRB and indicated proteins during microtubule disassembly (Left). Blebbistatin (Bleb, 10 µM) was pretreated for 2 hr in dNSpastin3Q group and followed by rapamycin treatment for 35 min. The fold change of cell rigidity was then measured by AFM (right). n = 16, 14, 14, and 14 cells in YFP-FKBP, dNSpastin3QED, dNSpastin3Q, and dNSpastin3Q+Bleb groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed with p values indicated.
An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice (2022)
Figure 1

Figure 1. Dynamics of experience-dependent parental behavior in virgin female mice and corresponding neuronal activity patterns.

A, Schematic representation of the pup retrieval test.

B, Total pup retrieval time in biological mothers (n=6), foster mothers (n=7), and virgin females (n=10) on 3 consecutive days. Data were analyzed using mixed model ANOVA with repeated measures and are displayed as mean ± s.e.m; **** significantly different from foster mothers, #### significantly different from mothers.

C,D Fraction of parental (C) and ignoring (D) behaviors of virgin females, of the total session duration in each of the three pup exposure days (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as individual measurements with the line connecting the mean value; *p < 0.05.

E, Schematic representation of a coronal section showing regions of the medial prefrontal cortex (mPFC).

F, A representative image of a coronal brain section immunostained for c-Fos after pup retrieval on day 1 and day 3. The mPFC region is indicated by dashed lines, which is also shown in the insets in higher magnification. Scale bar indicates 500 µm, 10× magnification.

G, Heat-map of the Z-score for the difference in the fraction of c-Fos expressing cells, between day 1 and day 3 of behavioral testing. Foster mothers (n=5), virgins with pups (n=6), virgins with object (n=7).

H, Representative coronal sections of the mPFC immunostained for c-Fos with the anatomical borders of the ACC indicated by dashed lines, in foster mothers (FM) and virgin females exposed to pups (V pups) or objects (V object) on day 1 and day 3 of behavioral testing. Scale bar 100 µm, 20× magnification.
An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice (2022)
Figure 3

Figure 3. Functional and molecular properties of the CL-ACC projection.

A, Schematic representation of the dual anterograde and retrograde tracing scheme for quantification of differential thalamic input to excitatory/inhibitory neurons in ACC.

B, Representative coronal sections of the PFC, following the labeling schemes described in (A), for mapping inputs onto ACC excitatory (B, top) and inhibitory (B, bottom) neurons. High magnification images (B, right top and bottom) show the double-labeled, putative starter cells for transsynaptic retrograde labeling. Scale bars represent 200 µm, 10× magnification (left top and bottom), and 50 µm (right top and bottom), 20× magnification.

C, Representative coronal images of the thalamus, immunolabeled for c-Fos, following retrograde labeling from excitatory (C, top) or inhibitory (C, bottom) neurons in the ACC. White arrows in high magnification images indicate colocalization of c-Fos and td-Tomato (C, right top and bottom). Scale bars represent 200 µm, 10× magnification (left top and bottom), and 25 µm (right top and bottom), 20× magnification.

D, Quantification of the number of neurons in CL region projecting to excitatory or inhibitory ACC neurons, as a fraction of the total thalamic contribution (top) and fraction of neurons co-expressing tdTomato and c-Fos of the total tdTomato+ population in CL (bottom). Foster mothers (n=3), virgins with pups (n=4), virgins with object (n=4). Data were analyzed using student's t-test and are displayed as mean ± s.e.m;

E, Representative in-situ hybridization images of Gal in the ILM taken from the Allen Mouse Brain Atlas, https://mouse.brain-map.org/experiment/ivt?id=70231997 (top left and right) and representative coronal images showing the expression of Gal neurons in Gal-Cre::FLEX-YFP female mice (bottom right and left). Scale bars represent 500 µm, 10× magnification (left bottom) and 200 µm (right bottom).

F, Representative image of the ILM immunolabeled for GFP and c-Fos, following pup retrieval test. Yellow and white arrows indicate c-Fos expressing Gal-positive and Gal-negative neurons, respectively. Scale bars represent 100 µm, 20× magnification.

G, Graph plots showing the fraction of c-Fos expressing Gal-positive (Galc-Fos+) and Gal-negative neurons (DAPIc-Fos+) in the CL, PCN, and CM after pup retrieval test (G, top) or object exposure (G, bottom). Data were analyzed using 2-way ANOVA and expressed as mean ± s.e.m.; n=3; *p < 0.05.
An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice (2022)
Figure 5

Figure 5. Acute chemogenetic activation/inhibition of the ACC affects the display of parental care behavior in virgin females.

A, Schematic representation of the chemocogenetic manipulation strategy in C57BL/6 mice.

B,C, Quantification of pup retrieval latencies following ACC chemogenetic activation with hM3D (n=6) (B) or chemogenetic inhibition using hM4D (n=7) (C) compared to control groups in virgin females (n=8). Data were analyzed using 2-way ANOVA with repeated measures and are displayed as mean ± s.e.m* p< 0.05, ** p< 0.01.

* significantly different between groups on day1. # significantly different between days within groups.

D, Same as (C) but for mothers (n=7)

E, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the PFC of virgin females, 2 hours following CNO/vehicle administration. Masks demonstrating distribution of DAPI + c-Fos (green), DAPI + dTomato (magenta) and their colocalization (white). Scale bars 100 µm, 20× magnification.

F, Fraction c-Fos+ cells of the total DAPI (top) or hM3D-2A-dTomato (bottom) population in the ACC, following CNO/vehicle administration. (n=3). Data were analyzed using student's t-test and are displayed as mean ± s.e.m; * p< 0.05, ** p< 0.01.

G, Representative images of the CL, immunolabeled for c-Fos, following hM3D-mediated neuronal activation of the ACC. Scale bars 200 µm, 20× magnification.

H, Spearman correlation between the hM3D-mediated activation of the ACC (as measured in (F, top)) and the CL region.
An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice (2022)
Figure 6

Figure 6. Acute chemogenetic activation CLGal+ neurons reduces pup retrieval latencies in virgin female mice.

A, Schematic representation of the chemogenetic manipulation strategy in Gal-Cre mice.

B, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the CL, 2 h after CNO administration. Scale bars 200 µm, 10× magnification (left) and 50 µm, 20× magnification (right).

C, Quantification of pup retrieval latencies in virgin females following CLGal+ chemogenetic activation with hM3D compared to GFP-expressing control group (n=7). Data were analyzed using 2-way ANOVA with repeated measures and are presented as mean ± s.e.m; * p < 0.05.
The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding (2020)
Figure 1.

Interaction of FGF2 and Spike RBD with immobilised heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. A Injection of 100 nM FGF2. B 64 nM S1 RBD protein. C 64 nM S1 RBD protein over a streptavidin surface (no heparin functionalization).
The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding (2020)
Figure 2.

The structural change of the SARS-CoV-2 S1 RBD observed in the presence of heparin by circular dichroism (CD) spectroscopy.(A) CD spectra of 200 μg/mL spike 1 alone (purple) or with 30 μg heparin (orange) in phosphate buffered saline pH 7.4. Theoretical sum of spike 1 alone and 30 μg of heparin (control) if no interaction was observed (black). (B) Δ secondary structure (%) of (A). A 2.4% increase in helix and 2.2% decrease in antiparallel secondary structural features were calculated (BestSel) for the observed spectrum compared to that of the theoretical, summative spectrum of the SARS-CoV-2 S1 RBD in the presence of heparin.
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 1

Figure 1. TBK1 kinase activity and C-terminal domain are required for restriction of S. Typhimurium replication.

A) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs stably expressing the indicated TBK1 alleles. Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points. Statistical analysis comparing Tbk1-/- MEFs and cells expressing TBK1 alleles. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.

B) Replication of S. Typhimurium in Atg5-/- MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 (10 nM) or DMSO. Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation (p.i.) following cell lysis.

C) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium. Percentage of S. Typhimurium coated with ubiquitin (detected by FK2 antibody) at 1 or 4 h p.i. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.

Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats (A-B). Mean and s.e.m. of three independent experiments with duplicate coverslips. >200 bacteria counted per coverslip (C). *p<0.05, ***p<0.001, one-way ANOVA with Dunnett’s multiple comparisons test.
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 2

Figure 2. TBK1 kinase activity and C-terminal adaptor binding domain are required to recruit WIPI1 and WIPI2 to S. Typhimurium.

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:LC3B (A), the indicated GFP:WIPI alleles (B), GFP:DFCP1 (FYVE* denotes a PI(3)-binding mutant of DFCP1) (D-E), or GFP:ML1N*2, a probe for PI(3,5)P2 (G). Where indicated, Wortmannin (Wort) was added at 100 nM. Confocal micrographs of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).

Mean and s.e.m. of at least three independent experiments with duplicate coverslips. >200 bacteria counted per coverslip. *p<0.05, **p<0.01 one-way ANOVA with Dunnett’s multiple comparisons test. Scale bar 10 μm.
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 3

Figure 3. WIPI2 restricts Salmonella proliferation

A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI1 (A) or WIPI2 (B). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. Western blot for GFP:WIPI1 and quantitative RT-PCR for WIPI2 upon the indicated siRNA treatments.

Mean and s.e.m. n=3 (A), n=6 (B). *p<0.05, Student’s t-test
Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2- dependent antibacterial autophagy (2016)
Figure 4

Figure 4. Recruitment of TBK1 to S. Typhimurium via NAP1 or NDP52, but not TANK, restricts bacterial proliferation and recruits WIPI1

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of four independent experiments. >200 bacteria counted per coverslip. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett’s multiple comparisons test.

C) TBK1-Nap1-NDP52 complex. N- and C-termini, domains and binding partners are indicated.