EMBO/soda-nel huggingface dataset:
Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing (2019)
Figure 2

Figure 2 Key features of the Nxph1-LNS2SS2- interface.

(A) View of the Nxph1-LNS2SS2- interface showing key LNS2 residues as sticks. The yellow surface represents the Nxph1 molecule.

(B) Sequence alignment of mouse Nrxn1-3 LNS2SS2- showing key regions involved in Nxph1 binding. Sequences are numbered in reference to the canonical UniProt mouse Nrxn1 sequence Q9CS84-1. Secondary structure elements of LNS2SS2- are shown at the top and correspond to elements shown in A.

(C) Electron density map showing the LNS2SS2- I401 residue (highlighted in red in B) interacting with a hydrophobic binding pocket formed primarily by Y249 and L251 of Nxph1.

(D) Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2- (I401Q) mutant compared to that of wild-type Nrxn1 LNS2SS2-. Replicate numbers are shown in E.

(E) Comparison of binding affinities for Nxph1 binding to mouse Nrxn1-3 LNS2SS2- and LNS2SS2- (I401Q), plotted on a logarithmic scale. Error bars represent SEM and replicate numbers are indicated in bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01.
Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing (2019)
Figure 3

Figure 3 Comparison of the Nxph1-LNS2SS2A+ structure to the Nxph1-LNS2SS2- structure and modulation of Nxph1-Nrxn1 LNS2 binding by alternative splicing.

(A) Overall view and close-up of superimposed Nxph1-LNS2SS2- and Nxph1-LNS2SS2A+ structures in cartoon representation.

(B) Electron density maps comparing the Nxph1-LNS2SS2- and Nxph1-LNS2SS2A+ structures. These maps cover the same region as shown in the close-up in A. The 2mFo-DFc map for the Nxph1-LNS2SS2A+ structure is shown in gray at a contour level of 1 σ. The positive and negative Fo-Fo difference density maps (the difference between Nxph1-LNS2SS2- and Nxph1-LNS2SS2A+ structure factor amplitudes using phases from the Nxph1-LNS2SS2A+ model) are shown in green and red, respectively, at a contour level of 3 σ.

(C) Sequence alignment of mouse Nrxn1-3 LNS2SS2A/AB+ showing key regions involved in Nxph1 binding and SS2A and SS2AB inserts. Sequences are numbered in reference to the canonical UniProt mouse Nrxn1 sequence Q9CS84-1. Secondary structure elements are shown at the top and correspond to elements shown in the Nxph1-LNS2SS2A+ structure in A.

(D) Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A+ and Nrxn1 LNS2SS2AB+ compared to that of Nrxn1 LNS2SS2-. Replicate numbers are shown in E.

(E) Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice-insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.
Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing (2019)
Figure 4

Figure 4 The SS2A insert does not affect Ca2+-sensitivity of Nxph1-LNS2 interactions. (A) Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added. Replicate numbers are shown in B.

(B) Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.
Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing (2019)
Figure 5

Figure 5 Isoform specificity of neurexin-neurexophilin interactions.

(A) Comparison of binding affinities of Nxph1 binding to different Nrxn1-3 LNS2 isoforms.

(B) Comparison of binding affinities for Nxph3 binding to Nrxn1-3 LNS2SS2-.

Error bars show SEM, and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, ****P < 0.0001.
ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells (2021)
Figure 1

Figure 1. Heterogenous exosome release from polarized MDCK cells.

A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.

B. PEG pellets prepared as in (A) were subjected to OptiPrep flotation analysis.

C. MDCK cells stably expressing human CD63 were cultured as in (A). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.

D. sEVs prepared as in (C) were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown.

E. Quantification of the NTA data obtained in five independent experiments.

F. MDCK cells were cultured as in (A). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.

G. sEVs prepared as in (F) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.

H. Quantification of the NTA data obtained in five independent experiments.

Data information: (A and B) Annexin I blots were separately obtained on different days using the same samples. (E and H) *P < 0.01 (two-sided Student's unpaired t test). Mean ± s.e.m. was shown.
ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells (2021)
Figure 2

Figure 2. GPRC5C is an apical-specific exosome protein.

A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining.

B. P100 prepared as in (A) were analyzed by LC-MS/MS. Venn diagrams represent the number of proteins detected in each sample with minimum three independent peptides. See also Table EV1-3.

C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated.

D. PEG pellets were subjected to OptiPrep flotation analysis.

E. MDCK cells were cultured as in (C). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.

F. MDCK cells stably expressing human CD63 were cultured as in (C). sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody. Note that GPRC5C was detected only in the apical sEV samples in all experiments performed.

Data information: (C and D) Annexin I blots were separately obtained on different days using the same samples.
ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells (2021)
Figure 3

Figure 3. ALIX, but not the ESCRT machinery, is required for apical exosome release.

A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.

B. The intensity of the bands shown in (A) was measured in three independent experiments.

C. sEVs prepared as in (A) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.

D. Quantification of the NTA data obtained in five independent experiments.

Data information: (A) CD63 blots were separately obtained on different days using the same samples. (B and D) *P < 0.05, **P < 0.01 (one-way ANOVA and Tukey's test). Mean ± s.e.m. was shown.
ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells (2021)
Figure 4

Fig. 4. ALIX and ceramide independently mediate apical and basolateral exosome release, respectively.

A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.

B. The intensity of the bands shown in (A) was measured in three independent experiments.

C. sEVs prepared as in (A) were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.

D. Quantification of the NTA data obtained in five independent experiments.

E. Working model of polarized sEV release from MDCK cells. Apical release of sEVs (mainly CD63-enriched, CD9-positive, Annexin I-negative exosomes) and basolateral release of sEVs (mainly CD9-enriched, CD63-positive, Annexin I-negative exosomes) are mediated by ALIX-Syntenin1-Syndecan1 and ceramide, respectively. Marker sizes on sEVs (colored circles) reflect the abundance of each exosome protein. Ceramide also contributes to apical release of uncharacterized "CD9/CD63-negative sEVs".

Data information: (A) CD63 blots were separately obtained on different days using the same samples. (B and D) *P < 0.01 (one-way ANOVA and Tukey's test). Mean ± s.e.m. was shown.

Expanded view figure legends
Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 1

Figure 1. Primary photoreceptors release EVs that exert their function in Müller glial cells but not photoreceptors.

a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right;

b, Representative SEM microphotograph of cultured P8 Nrl.Gfp+/+ photoreceptors; arrows indicate membrane budding of processes or EVs; dashed circles denote EVs; Scale bar = 3µm;

c, Representative TEM microphotograph of 100K EV pellet derived from Nrl.Gfp+/+ photoreceptor cultures (N = 10 independent preparations); Scale bar = 100nm;

d & e, Representative Dynamic Light Scatter (DLS) plot of 100K EV sample (N = 13 samples), showing (d) average intensity and (e) volume against diameter (13 DLS measurements per sample);

f, Representative Dot Blot of 100K EV pellet (each dot represents 3 pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin;

g, RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/-) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples;

h, schematic representation of Cre-LoxP system to assess EV function in co-culture of Nrl.Cre+/- photoreceptors (PRs) with mTmGfloxed reporter retinal cells in non-proximity (transwell) versus physical proximity (contact);

i, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test

j, representative maximum intensity projection (MIP) confocal images of mTmGfloxed reporter retinal cells, (left) treated with AAV-Nrl.Cre virus (positive control), (middle) co-culture with Nrl.Cre+/- photoreceptors separated by transwell (non-proximity) or (right) in contact; Scale bar = 20µm; Red = myrRFP-expressing mTmGfloxed reporter, no recombination; green = myrGFP-expressing mTmGfloxed reporter, cells recombined upon acquiring Cre; blue = nuclei. N = 7 cultures.

Data information: Graphs show mean ± S.D.
Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 2

Figure 2: Nrl.Cre+/- photoreceptor-derived EVs are taken up by Müller glia cells when injected in vivo.

a, Schematic representation of photoreceptor transplantation, shown in (b);

b, (left) P8 Nrl.Cre+/- photoreceptors transplanted into subretinal space of TdTomatofloxed reporter mice (N = 5 eyes), compared to (middle) subretinal injection of AAV-Nrl.Cre subretinal injection (10^12 vp/ml), (N = 10 eyes) and (right) contralateral uninjected control (N = 10 eyes). Arrows indicate recombined host photoreceptors (white arrows) or RPE (green arrows). Note clearly recombined RPE cells upon AAV transduction.

c, schematic representation of subretinal EV injection, shown in (d). Red indicates host cells undergoing Cre-mediated recombination and expressing TdTomato (red);

d, P8 Nrl.Cre+/- PR -derived (left) 100K, (middle) 10K and (right) 2K EV pellets injected into the subretinal space of TdTomatofloxed reporter mice (N = 4 eyes per EV preparation). Recombination was restricted to Müller Glia (asterisks).

e, schematic representation of intravitreal EV injection, shown in (f);

f, (left) intravitreal injection of P8 Nrl.Cre+/- photoreceptor-derived 100K EV pellets (N = 2 eyes). Recombination was restricted to Müller Glia (asterisks)with little or no recombination in either photoreceptors or the RPE; (right) subretinal injection of recombinant Cre protein (control) into TdTomatofloxed reporter mice. Blue = nuclei, red = TdTomato+ve recombined cells. N = 4 retinae per group. Representative MIP confocal images;

g, Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/- P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± S.D.

Data information: Scale bars = 50µm; PR - Photoreceptor; MG - Müller Glia; RPE - Retinal Pigment Epithelium; ONL - outer nuclear layer; INL - inner nuclear layer; GCL - Ganglion Cell Layer.
Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 3

Figure 3: Photoreceptors form nanotube-like processes to connect with neighbouring photoreceptors in culture

a-d, Representative 3D deconvolved MIP images from live imaging of Nrl.Gfp+/+ (green) P8 photoreceptors at DIV1-3, labelled with SiR-actin (red) (N = 7 independent cultures). Photoreceptors exhibit a variety of processes, including (a) neurites (blue arrow), (b) nascent inner segment-like protrusions (yellow arrow), and (c) short and (d) long (magenta arrows) actin+ve nanotube-like (herein termed PhNTs) connections; Scale bar = 5 µm;

e-g, Representative 3D deconvolved MIP images from live imaging of Nrl.Gfp+/+ (green) P8 photoreceptors at DIV1-3 labelled with SiR-tubulin (red) (N = 7 independent cultures). Photoreceptors exhibit a variety of processes, including nascent inner segment-like protrusions (yellow arrows), neurites (blue arrows) and rare tubulin+ve PhNTs (magenta arrows); Scale bar = 5 µm;

h, Representative SEM images with digitally enhanced microphotographs of cultured P8 Nrl.Gfp+/+ photoreceptors. Red arrows indicate PhNT connections between neighbouring photoreceptors, while blue arrows indicate broken PhNT connections (N = 3 cultures). Scale bar = 5 µm;

i & j, Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (i) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (j), a free-floating PhNT (magenta arrow). Scale bar = 10 µm;

k & l, Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1096 cells, nneurites = 440, nnanotubes = 31 where (k) neurites. P values = **** p < 0.0001 (bottom vs top); **p = 0.002 (bottom vs middle); **p = 0.003 (middle vs top) and (l) PhNTs; P-values = **** p < 0.0001 (bottom vs middle & middle vs top) . One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test shows means vary significantly.

m & n, Diameter and length quantification of SiR-actin+ve PhNTs (N = 7 cultures; ncells = 646 cells; nnanotubes = 35, red dots) compared to SiR-tubulin+ve PhNTs (N = 7 cultures; ncells = 885 cells, nnanotubes = 15; blue dots). One way ANOVA, non-parametric, Kolmogorov-Smirnov test shows non-significant (ns) difference in length but significant difference in diameter between actin+ve versus tubulin+ve PhNTs. P values; ** p = 0.001, and p = 0.245, in (m) and (n), respectively.

Data information: Graphs show mean ± S.D.
Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 4

Figure 4: PhNTs facilitate recovery after photobleaching in culture.

a, Schematic representation of cytoplasmic (c)GFP mobility during fluorescence recovery after photobleaching (FRAP).

b, FRAP recovery curves for PhNT-connected cells (green, n = 42, R2 = 0.99), clustered cells (black, n = 16, R2 = 0.94) and single cells (red, n = 46, R2 = 0.97); Mann-Whitney analysis of fit for curves.

c, FRAP recovery curves in un-bleached reference cells (black, n = 41) versus "donor" PhNT-connected photoreceptor cells (purple, n = 46) show significant slope difference. Mann-Whitney analysis of fit for curves, *** p<0.001;

d, representative time lapse images of cGFP mobility between two PhNT-connected Nrl.Gfp+/+ photoreceptors (dashed ellipse = bleached area) versus an isolated cell (solid ellipse = bleached area) and clustered cells (dot ellipse = bleached area). Images show pre-bleach (t = -5''), bleach (t = 0''), and post-bleach (t = 35'' and 215''). Unbleached reference cells also indicated (arrows); Green = cytoplasm; Scale bar = 5µm.
Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 5

Figure 5: PhNTs enable the intercellular transfer of cytoplasmic and membrane reporters and, rarely, organelles, in culture.

a, Flow cytometry analysis of cGFP and cDsRed following co-culture of Nrl.Gfp and DsRed cells (1:1) and analyzed at 3 DIC (N = 3 independent co-cultures with a technical triplicate), 7 DIC (N = 5), 21 DIC (N = 4); One-way ANOVA non-parametric, Kruskal Wallis;

b, flow cytometry analysis of DiI/DiO dual labelling of CD73+ cells following co-culture of DiI- and DiO-labelled photoreceptors (1:1) with pharmacological interventions applied at 5 DIC for 48hrs, including vehicle control (DMSO), Cytochalasin D (2 μM) and Latrunculin B (5 μM), after wash-off of Cyto D and LatrB, and a final wash control for DiI/DiO carry over; N = 4 independent co-cultures per condition; One-way ANOVA non-parametric, F-test, Kolmogorov-Smirnov;

c, Representative MIP of DiI/DiO co-cultures after fixation, showing DiO+ve cell (A) and a DiI+ve cell (B) cell connected by a PhNT. Note DiI and DiO puncta in the respective acceptor cells. Blue = Dapi (nuclei), Green = DiO, Red = DiI. Scale bar = 5 µm;

d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell "A" and a PhNT connecting cells "A" and "B"; Scale bar = 2 µm;

e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.

Data information: Graphs show mean ± S.D.
Nanotube-like processes facilitate material transfer between photoreceptors (2021)
Figure 7

Figure 7: Generation of Nrl.Cre+/-/TdTomatofloxed aggregate chimeras reveals intercellular exchange of Cre between photoreceptors in vivo.

a, Schematic representation of Nrl.Cre+/-/TdTomato+/- floxed chimera generation via morula aggregation, which results in chimeric mosaicism in the neural retina. Recombination and expression of TdTomato is only possible if Nrl.Cre+/- photoreceptors exchange material (Cre) with TdTomatofloxed cells via intercellular communication;

b, Representative tile scan of un-injected Nrl.Cre +/-/TdTomato+/- chimera showing endogenous recombination (white); ROIs, as indicated with dashed or dot boxes; Scale bars = 50 µm;

c & d, ROI from un-injected Nrl.Cre +/-/TdTomato+/- chimera showing spontaneous recombination presenting in mosaic stripe patterning; Scale bar = 50 µm in c and 10 µm in d;

e & f, Representative images of Nrl.Cre+/-/TdTomato+/- chimera after receiving a subretinal injection of AAV-Nrl.Cre. The injected eyes show widespread recombination, which also shows the anticipated mosaic stripe patterning; Scale bar = 50 µm in (e) and 10 µm in (f);

g, Quantification of number of recombined cells in un-injected (29,825 cells ± 2031) and AAV-Nrl.Cre-injected (131,562 ± 8478) Nrl-Cre+/-/TdTomato+/- chimeras, compared with un-injected Nrl-Cre-/-(wt)/TdTomato+/- chimera (0 ± 0). Graph shows mean ± SD

Data information: white = TdTomato+ve recombined cells; blue = nuclei. RPE- Retinal Pigment Epithelium; ONL - outer nuclear layer; INL - inner nuclear layer; GCL- Ganglion Cell Layer.

Expanded View Figure Legends
The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel (2008)
figf1

a, A TRPML1Va-expressing HEK293T cell showed a large inwardly rectifying whole-cell current elicited by voltage steps (from -140 mV to +80 mV in increments of 20 mV) in the standard extracellular (Tyrode’s) bath solution. Step duration, 90 ms; holding potential, 0 mV; Vm, membrane potential. b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution. c, Inwardly rectifying step currents were evoked by 30 mM Fe2+ solution (pH 4.6) in the same cell as shown in a and b. d, pH-dependence of IFe/TRPML1Va. Whole-cell currents were elicited by repeated voltage ramps (-100 to +100 mV; 400 ms) with a 4-s interval between ramps. Only a portion of the voltage protocol is shown. Holding potential, 0 mV. e, Large IFe/TRPML2Va was seen in the presence of 30 and 105 mM Fe2+ (pH 4.6). f, Little or no IFe was seen in a TRPML3Va-expressing cell.