Journal of Cell Biology

Journal of Cell Biology (JCB)

Published by Rockefeller University Press

Online ISSN: 1540-8140

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Print ISSN: 0021-9525

Disciplines: Cell Biology, Molecular Biology and Genetics, Immunology, Neurobiology, Metabolism, Microbiology, Developmental Biology

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IQGAP1 plays a role in autophagosome closure. (A) Scatter plot of ATG9A proximity proteome comparing starvation-induced autophagy (EBSS, 90 min) and treatment with CCCP (20 µM, 6 h) identified by LC-MS/MS in FLAG-APEX2-ATG9A Flp-In T-REx HEK293(TetON) cells. For highlighted proteins (colors defined in inset legend), functions are separated by the stage of autophagosome biogenesis. (B) Schematic ATG9A was modified from the AlphaFold entry AF-Q7Z3C6 by rotating the unstructured C-terminal loop to avoid clashes with the membrane. Successive stages of autophagosome biogenesis: initiation (X), expansion (Y), and closure (Z). (C) Schematic representation of quantitative high content microscopy HaloTag (HT)-LC3B based closure assay (MIL/MPL HCM) encompassing incubation with membrane-impermeant HT ligand (MIL) to stain and saturate HT-LC3B-II accessible to the cytosol followed by membrane-permeant HT ligand (MPL) to stain LC3B-II (protected from and free of MIL because of sequestration within sealed membranes). (D) IQGAP1 knockdown (siRNA pool) in Huh7 HT-LC3B cells, immunoblot analysis. (E) MIL/MPL HCM quantification in Huh7 HT-LC3B control cells or cells knocked down for IQGAP1. Starvation in EBSS, 90 min incubation ± 100 nM BafA1. (i) MPL⁺ puncta (red symbols), closed autophagosomes. (ii) MIL⁺ puncta (green symbols), unclosed phagophores, and other accessible HT-LC3B; (iii) Ratio of MIL⁺ and MPL⁺ profiles (puncta/cell; gray symbols) in i and ii. Circles, siRNA control cells; squares, cells knocked down for IQGAP1. (F) Immunoblot of IQGAP1 KD with individual siRNAs, MIL/MPL HCM quantification in Huh7 HT-LC3B control cells or cells knocked down for IQGAP1 (siRNA1, squares; siRNA2, triangles) and complementation with siRNA resistant constructs pDest-3xFLAG-iQGAP1Res1 (diamonds) or pDest-3xFLAG-IQGAP1Res2 (inverted triangles) against siRNA1 and siRNA2, respectively. Starvation in EBSS, 90 min incubation ± 100 nM BafA1. (i–iii) MPL⁺ puncta (red symbols), (ii) MIL⁺ puncta (green symbols), (iii) Ratio of MIL⁺ and MPL⁺ profiles (puncta/cell; gray symbols). HCM parameters: 60 fields/well, >500 primary objects (cells)/well; 6 (E) or 4 (F) wells per sample/plate. Statistical significance was determined by one-way ANOVA and post-hoc Tukey’s multiple comparison test. Data, means ± SD, n = 5 (E) or 3 (F) biologically independent experiments per condition. Source data are available for this figure: SourceData F1.
IQGAP1 is necessary for degradative autophagy and bridges ATG9A with CHMP2A. (A) Schematic, TMRHT release assay. TMRHT-LC3B (HaloTag-LC3B) is processed by lysosomal hydrolases releasing the TMRHT fragment from a fusion with LC3B. Released (HaloTag stabilized by TMR) is detectable by in-gel fluorescence and immunoblotting. Top, open phagophores do not yield the TMRHT fragment. Bottom, closed autophagosomes fuse with lysosomes and the TMRHT fragment is released. (B)TMRHT release in IQGAP1 knockdown or control (siScr) cells stably expressing HT-LC3B. TMR+, cells incubated with TMR for 30 min. Cells were starved in EBSS for 90 min, lysed, and processed for in-gel fluorescence and immunoblotting. (i) In-gel fluorescence detection of released TMRHT. (ii) Immunoblot detection of released TMRHT. (iii) quantification of released TMRHT in immunoblots. (C) ESCRT protein subcomplexes with components present in or absent from LC-MS/MS after proximity biotinylation with APEX2-ATG9A. Cells (FLAG-APEX2-ATG9A Flp-In T-REx HEK293[TetON]) were incubated in EBSS (90 min) or treated with CCCP in full medium for 6 h. Black, proteins detected in all conditions; blue, detected only in EBSS; purple, detected only in CCCP; red, not detected in any samples. Note ESCRTs absent from proteomic dataset (red color). (D) Co-IP analysis of GFP-ATG9A with endogenous CHMP2A in Huh7 cells, control (siScr) or knocked down for IQGAP1 by siRNA. Cells were treated with protonophore CCCP for 6 h as a means to collapse organellar proton gradients. (i) Immunoblot, IQGAP1 knockdown in Huh7 cells. (ii) Western blot, Co-IP analysis of GFP-ATG9A (GFP pulldown), and endogenous CHMP2A in control and IQGAP1 depleted cells. (iii) Quantification of Co-IP analyses (CHMP2A band intensity was ratioed to the intensity of the upper band in GFP-ATG9A blots). Data, means ± SD, n = 3 ANOVA. (E) Summary of findings in Fig. 2. Source data are available for this figure: SourceData F2.
ATG9A colocalizes with CHMP2A and supports sequential stages in autophagosome biogenesis. (A) Confocal microscopy imaging of Huh7WT cells transiently transfected with pDest-3xFLAG-ATG9A and GFP-CHMP2A and stained for endogenous LC3B. Cells were starved in EBSS for 90 min. White square, enlarged area in the inset (merged image; dashed diagonal line - section). (B) Profile intensity (dashed diagonal in A inset) for multiple fluorescence channels. (C) Immunoblot analysis of ATG9A KO in Huh7 cells and MIL/MPL HCM closure assay in Huh7WT (circles) and Huh7ATG9AKO (squares) cells stably expressing HT-LC3B; starvation-induced autophagy (EBSS, 90 min) ± 100 nM BafA1. (i–iii) Quantifications: (i) MPL⁺ HT-LC3B (red symbols); (ii) MIL⁺ HT-LC3B (green symbols); (iii) MIL/MPL ratios (puncta/cell; gray symbols). (D) HCM images: red masks, MPL⁺ profiles, green masks, MIL⁺ profiles. Quantification: >500 (cells)/well with 80 fields/well; 6 wells per sample/plate. Data, means ± SD, n = 5 (biologically independent experiments); one-way ANOVA followed by Tukey’s multiple comparison test. (E) Complementation analysis of Huh7ATG9AKO HT-LC3B cells transfected with pDest-3xFLAG, pDest-3xFLAG-ATG9AWT or pDest-3xFLAG-ATG9AM33 mutant. Source data are available for this figure: SourceData F3.
In vitro assay for autophagosome closure. (A) SolVit (sealing of organellar limiting membranes in vitro) assay schematic: in vitro complementation by mixing postnuclear supernatants (PNS) from ATG9AKO HT-LC3B cells (Acceptor) with PNS from ATG9AWT or ATG9AKO cells (Donor), ±ATP, incubated for 1 h. PNS were from cells treated with 20 μM CCCP for 6 h. Reaction products were stained with MIL and MPL sequentially and immobilized in mounting media on the bottom of 96-well plates followed by HCM quantification. (B i–iii) MPL⁺ profiles (red); (ii) MIL⁺ profiles (green). (iii) MIL/MPL ratios (gray). Each HCM experimental point: 1,000 valid primary objects/cells per well, 5 wells/sample. Data, means ± SD, n = 3 (biologically independent experiments); one-way ANOVA followed by Tukey’s multiple comparison test. (C) Examples of HCM images from SolVit assay. Red profiles, MPL⁺ closed LCB⁺ membranes; Green profiles, MIL⁺ unclosed LC3B⁺ membranes. Scale bars, 3 μm. Source data are available for this figure: SourceData F4.
Ultrastructural analysis of ATG9A, IQGAP1, and CHMP2A in autophagosome closure. (A–C) Representative transmission electron microscopy (EM) micrographs of profiles in HeLaWT and HeLaATG9AKO (A), and HeLa cells treated with siRNA control (siScr), CHMP2A siRNA, or IQGAP1 siRNA (B and C). Cells were treated with EBSS (90 min) to induce autophagy. Examples of mitochondria engulfed in phagophores (C) in CHMP2A and IQGAP1 knockdown cells after EBSS treatment (90 min). (D) Quantification of autophagic structures (average number/cell section) in cells treated with EBSS (90 min) 60 sections were examined for counting. A, autophagosomes (engulfed content of similar electron density to surrounding cytosol); ER, endoplasmic reticulum; HDP, high density particle; M, mitochondria; P, phagophores. Statistics, unpaired t test. Data, sample mean, SE. Source data are available for this figure: SourceData F5.

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ATG9A facilitates the closure of mammalian autophagosomes

January 2025

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183 Reads

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Aims and scope


Journal of Cell Biology (JCB) publishes advances in any area of basic cell biology as well as applied cellular advances in fields such as immunology, neurobiology, metabolism, microbiology, developmental biology, and plant biology. Est. 1955

Recent articles


Prickle2 regulates apical junction remodeling and tissue fluidity during vertebrate neurulation
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February 2025

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15 Reads

The process of folding the flat neuroectoderm into an elongated neural tube depends on tissue fluidity, a property that allows epithelial deformation while preserving tissue integrity. Neural tube folding also requires the planar cell polarity (PCP) pathway. Here, we report that Prickle2 (Pk2), a core PCP component, increases tissue fluidity by promoting the remodeling of apical junctions (AJs) in Xenopus embryos. This Pk2 activity is mediated by the unique evolutionarily conserved Ser/Thr-rich region (STR) in the carboxyterminal half of the protein. Mechanistically, the effects of Pk2 require Rac1 and are accompanied by increased dynamics of C-cadherin and tricellular junctions, the hotspots of AJ remodeling. Notably, Pk2 depletion leads to the accumulation of mediolaterally oriented cells in the neuroectoderm, whereas the overexpression of Pk2 or Pk1 containing the Pk2-derived STR promotes cell elongation along the anteroposterior axis. We propose that Pk2-dependent regulation of tissue fluidity contributes to anteroposterior tissue elongation in response to extrinsic cues.


An S-acylated N-terminus and a conserved loop regulate the activity of the ABHD17 deacylase

February 2025

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12 Reads

The dynamic addition and removal of long-chain fatty acids modulate protein function and localization. The α/β hydrolase domain–containing (ABHD) 17 enzymes remove acyl chains from membrane-localized proteins such as the oncoprotein NRas, but how the ABHD17 proteins are regulated is unknown. Here, we used cell-based studies and molecular dynamics simulations to show that ABHD17 activity is controlled by two mobile elements—an S-acylated N-terminal helix and a loop—that flank the putative substrate-binding pocket. Multiple S-acylation events anchor the N-terminal helix in the membrane, enabling hydrophobic residues in the loop to engage with the bilayer. This stabilizes the conformation of both helix and loop, alters the conformation of the binding pocket, and optimally positions the enzyme for substrate engagement. S-acylation may be a general feature of acyl-protein thioesterases. By providing a mechanistic understanding of how the lipid modification of a lipid-removing enzyme promotes its enzymatic activity, this work contributes to our understanding of cellular S-acylation cycles.



NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

February 2025

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22 Reads

Nuclear mitotic apparatus protein (NuMA) is indispensable for the mitotic functions of the major microtubule minus-end directed motor cytoplasmic dynein 1. NuMA and dynein are both essential for correct spindle pole organization. How these proteins cooperate to gather microtubule minus ends at spindle poles remains unclear. Here, we use microscopy-based in vitro reconstitutions to demonstrate that NuMA is a dynein adaptor, activating processive dynein motility together with dynein’s cofactors dynactin and Lissencephaly-1 (Lis1). Additionally, we find that NuMA binds and stabilizes microtubule minus ends, allowing dynein/dynactin/NuMA to transport microtubule minus ends as cargo to other minus ends. We further show that the microtubule-nucleating γ-tubulin ring complex (γTuRC) hinders NuMA binding and that NuMA only caps minus ends of γTuRC-nucleated microtubules after γTuRC release. These results provide new mechanistic insight into how dynein, dynactin, NuMA, and Lis1 together with γTuRC and uncapping proteins cooperate to organize spindle poles in cells.


Single cell analysis reveals profound inhibition of AKT by PH-AKT PIP3 biosensor. (A) AKT is autoinhibited by its PIP3 binding PH domain. PIP3 production alleviates this steric inhibition, facilitating activating phosphorylations at S473 and T308. Over-expression of PH-AKT is hypothesized to out-compete the endogenous AKT’s PH domain. (B) Inhibition of AKT S473 phosphorylation is not apparent at the population level. HEK293A cells were serum starved and then treated (where indicated) with 10 ng/ml EGF to activate PI3K. After 5 min, cells were lyzed and analyzed by western blot for total Akt and Akt-pS473. (C) Quantification of blots from three experiments like that shown in B. Data are means ± s.e. (D–F) Inhibition of Akt activation (via S473 phosphorylation) is apparent through analysis of single cells. HEK293A cells were serum starved (D) or stimulated with EGF (E), then fixed and stained with antibodies against pS473 along with DAPI for nuclear DNA (cyan). (D and E) Images show high-resolution confocal micrographs (1.45 NA 100× oil-immersion objective) of representative cells, whereas (F) shows mean fluorescence intensity measurements of cells imaged at low resolution (0.75 NA 20× air objective) to capture fluorescence from the entire volume of the cells. Graphs show medians ± 95% confidence interval of the median from 82 to 160 cells pooled from three experiments (medians are reported since the data are not normally distributed). Results of a multiple comparisons post-hoc test (ns = P > 0.99; **** = P < 0.0001) are indicated from a Kruskal-Wallis test (Kruskall-Wallis statistic = 839.2, six groups, P < 0.0001). Source data are available for this figure: SourceData F1.
Fluorescent tagging of AKT1 at its genomic locus. (A) CRISPR/Cas9 directed cutting at the 3′ end of the AKT1 ORF is coupled to homology-directed repair to integrate an in-frame NG2¹¹ tag, encoding the 11th strand of neonGreen2. The edit is made in a HEK293A cell line stably expressing NG21–10, the remainder of the neonGreen protein. The two neonGreen2 protein fragments assemble in the cell to generate fluorescent NG2 protein. (B) Western blot of AKT protein from a sorted, polyclonal population of edited cells shows the appearance of a second molecular weight band, consistent with the 81.9 kDa complex between AKT1 (55.7 kDa) and NG2 (26.2 kDa). (C) TIRF imaging of AKT1-NG2 cells exhibit discrete fluorescent spots at the cell surface, which increase in density after PI3K activation with 10 ng/ml EGF. The light lines are data points from images recorded at 20 Hz; the thicker, darker line is the 5 s rolling average. (D and E) AKT1-NG2 spots are single molecules: (D) The intensity of fluorescent AKT1 spots pooled from a representative experiment shows a mono-modal lognormal distribution (green). The data are fit with a model assuming the intensity is derived from a mixture of monomeric, dimeric or trimeric fluorescent proteins calibrated against a known monomeric mNeonGreen fluorescent protein distribution. The fit predicts 98.1% monomers with a reduced χ² of 1.06. (E) The results of this analysis pooled across six cells from three independent experiments yields consistent results with mean χ² of 1.67 ± 0.40 (s.e.). (F) Extended TIRF imaging of AKT-NG2 cells with reduced duty cycle to minimize photobleaching reveals robust recruitment of AKT1 after EGF stimulation. Data are means ± s.e. of 20 cells pooled from two independent experiments. (G) PI3K activation is sufficient to recruit AKT1 to the plasma membrane. AKT1-NG2 cells were transfected with PM-targeted Lyn N-terminal 11 residues fused to FRB and the PI3K p110 catalytic subunit-binding iSH2 fused to FKBP and mCherry. 1 µM rapamycin was added to cells to induce dimerization of FRB and FKBP and hence recruitment of iSH2/p110 to the plasma membrane, inducing PIP3 synthesis. AKT1-NG2 is further increased on the PM by this maneuver. Data are means ± s.e. of 32 cells pooled from three independent experiments. Insets in F and G show zoomed view indicated in the center of the cell. Source data are available for this figure: SourceData F2.
PH-AKT1 PIP3 biosensor abolishes endogenous AKT recruitment to the PM. (A) Hypothesized competition for PIP3 between the overexpressed PH domain and endogenous AKT1-NG2. (B) Raw 16-bit intensity levels of mCherry fluorescence in HEK293A cells transiently transfected with the indicated mass of PH-AKT1-mCherry for 24 h. Small data points represent measurements of individual cells, whereas large points are the means of each of three independent experiments. Points are color matched by experiment. Grand means ± s.e. are also indicated. (C) TIRF imaging of AKT1-NG2 cells from (B) stimulated with 10 ng/ml EGF. Insets show zoomed view indicated in the center of the cell. (D) Relative PM fluorescence of PH-AKT1-mCherry during time lapse imaging and stimulation with 10 ng/ml EGF. (E) As in D, except the density of endogenous AKT1-NG2 molecules are counted. Data in D and E are grand means of three experiments ± s.e. imaging 8–10 cell each. (F) Data from E were pooled from all three experiments, and raw 12-bit mCherry fluorescence intensity data (constant laser power and camera gain between experiments) was used to bin cellular measurements as indicated on the x-axis. Small points represent individual cell measurements, whereas the large symbols are means ± 95% confidence interval. Data were normal by Kolmogorov-Smirnov test, and results of a Holm-Šídák’s multiple comparisons test are indicated comparing to control cells (“0” cohort); *** P = 0.002, **** P < 0.0001, performed post-hoc to an ordinary one-way ANOVA (F = 36.73, P < 0.0001).
PIP3 and PI(3,4)P2 biosensors inhibit endogenous AKT recruitment to the PM. (A) Hypothesized competition for PIP3 between PIP3 biosensors and endogenous AKT1-NG2. (B) AKT-NG2 cells were transiently transfected with the indicated PIP3 biosensors, or pUC19 as an inert control, and imaged after 24 h by TIRFM. Data quantify AKT-NG2 density at the cell surface. The time-lapse data are grand means ± s.e. of 3–4 independent experiments imaging 9–10 cells each. (C) The scatter plots show individual experiment means (small symbols) and grand means ± s.e. (D) As in A, except high and low avidity versions of a PI(3,4)P2-selective biosensor were tested. (E) Data are grand means ± s.e. of 3–5 independent experiments imaging 6–10 cells each. (F) summary data for E as described in C. Results of a Tukey’s multiple comparisons test are indicated comparing to pUC19 cells * P < 0.05, ** P < 0.005, performed post-hoc to an ordinary one-way ANOVA (C: F = 12.55, P = 0.0010; F: F =4.702, P = 0.0363).
Weak expression of PIP3 and PI(3,4)P2 biosensors does not inhibit AKT1 translocation, and reveals improved dynamic range and kinetic fidelity. (A) Hypothesized weak expression of biosensors that do not sequester a large fraction of the available lipids. (B) Example TIRFM images of AKT1-NG2 cells transiently transfected with aPHx1 PIP3 or cPHx1 PI(3,4)P2 probes for 2–4 h before imaging and stimulation with EGF. Note the single molecule expression of the biosensors. Insets show zoomed view indicated in the center of the cell. (C) Quantification of AKT1-NG2 translocation of aPHx1, cPHx1 or pUC19 (control)-transfected cells, showing no effect. The scatter plot shows individual experiment means (small symbols) and grand means ± s.e. P values of a Tukey’s multiple comparisons test are indicated comparing to pUC19 cells, performed post-hoc to an ordinary one-way ANOVA (F = 0.8206, P = 0.4784). (D) Quantification of the lipid biosensor density at the cell surface from the same experiments. (E) As in D, except data were normalized to the maximum density for each experiment, showing the characteristic lagging accumulation of PI(3,4)P2 versus PIP3. (F–I) Comparison of weakly (single-molecule level) expressed biosensors with the same constructs strongly over-expressed (24 h). (F) comparison of the change in fluorescence or molecule density versus baseline for weakly and strongly expressed PIP3 biosensor, aPHx1. (G) as in F, but data are normalized to the maximum for each experiment. (H) comparison of the change in fluorescence or molecule density versus baseline for weakly and strongly expressed PI(3,4)P2 biosensor, cPHx1. (I) As in I, but data are normalized to the maximum for each experiment. Data are grand means ± s.e. of 3–4 experiments analyzing 9–10 cells each. The data for the over-expressed biosensor were generated from the same experiment as presented in Fig. 4. Solid lines in G and I show fits to two co-operative reactions (synthesis and degradation). See text for details.
Single-molecule lipid biosensors mitigate inhibition of endogenous effector proteins

February 2025

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5 Reads

Genetically encoded lipid biosensors uniquely provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools have significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with effectors, potentially inhibiting signaling. Here, we show that although PI 3-kinase (PI3K)–mediated activation of AKT is not significantly reduced in a cell population transfected with a PH-AKT1 PIP3/PI(3,4)P2 biosensor, single cells expressing PH-AKT at visible levels have reduced activation. Tagging endogenous AKT1 with neonGreen reveals its EGF-mediated translocation to the plasma membrane. Co-transfection with the PH-AKT1 or other PIP3 biosensors eliminates this translocation, despite robust recruitment of the biosensors. Inhibition is even observed with PI(3,4)P2-selective biosensor. However, expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with overexpressed biosensor. This approach represents a noninvasive way to probe spatiotemporal dynamics of PI3K signaling in living cells.


When one nucleus is not enough: Intestinal polyploidy fuels healthier progeny in C. elegans

February 2025

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1 Read

In this issue, Lessenger and colleagues (https://doi.org/10.1083/jcb.202403154) investigate why certain differentiated tissues require extremely high DNA content. Using the nematode worm Caenorhabditis elegans, they show that restricting genome copies in intestinal cells triggers compensatory gene expression adaptations, which maintain organismal fitness at the expense of offspring vitality.


Structure of the F-tractin–F-actin complex

February 2025

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28 Reads

F-tractin is a peptide widely used to visualize the actin cytoskeleton in live eukaryotic cells but has been reported to impair cell migration and induce actin bundling at high expression levels. To elucidate these effects, we determined the cryo-EM structure of the F-tractin–F-actin complex, revealing that F-tractin consists of a flexible N-terminal region and an amphipathic C-terminal helix. The N-terminal part is dispensable for F-actin binding but responsible for the bundling effect. Based on these insights, we developed an optimized F-tractin, which eliminates the N-terminal region and minimizes bundling while retaining strong actin labeling. The C-terminal helix interacts with a hydrophobic pocket formed by two neighboring actin subunits, an interaction region shared by many actin-binding polypeptides, including the popular actin-binding probe Lifeact. Thus, rather than contrasting F-tractin and Lifeact, our data indicate that these peptides have analogous modes of interaction with F-actin. Our study dissects the structural elements of F-tractin and provides a foundation for developing future actin probes.


The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux

February 2025

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11 Reads

During autophagy, toxic cargo is encapsulated by autophagosomes and trafficked to lysosomes for degradation. NBR1, an autophagy receptor targeting ubiquitinated aggregates, serves as a model for studying the multivalent, heterotypic interactions of cargo-bound receptors. Here, we find that three critical NBR1 partners—ATG8-family proteins, FIP200, and TAX1BP1—each bind to distinct, overlapping determinants within a short linear interaction motif (SLiM). To explore whether overlapping SLiMs extend beyond NBR1, we analyzed >100 LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Phosphomimetic peptides demonstrate that phosphorylation generally enhances FIP200 and ATG8-family binding but not TAX1BP1, indicating differential regulation. In vivo, LIR-mediated interactions with TAX1BP1 promote optimal NBR1 flux by leveraging additional functionalities from TAX1BP1. These findings reveal a one-to-many binding modality in the LIR motif of NBR1, illustrating the cooperative mechanisms of autophagy receptors and the regulatory potential of multifunctional SLiMs.


VPS41 recruits biosynthetic LAMP-positive vesicles through interaction with Arl8b

February 2025

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29 Reads

Vacuolar protein sorting 41 (VPS41), a component of the homotypic fusion and protein sorting (HOPS) complex for lysosomal fusion, is essential for the trafficking of lysosomal membrane proteins via lysosome-associated membrane protein (LAMP) carriers from the trans-Golgi network (TGN) to endo/lysosomes. However, the molecular mechanisms underlying this pathway and VPS41’s role herein remain poorly understood. Here, we investigated the effects of ectopically localizing VPS41 to mitochondria on LAMP distribution. Using electron microscopy, we identified that mitochondrial-localized VPS41 recruited LAMP1- and LAMP2A-positive vesicles resembling LAMP carriers. The retention using selective hooks (RUSH) system further revealed that newly synthesized LAMPs were specifically recruited by mitochondrial VPS41, a function not shared by other HOPS subunits. Notably, we identified the small GTPase Arl8b as a critical factor for LAMP carrier trafficking. Arl8b was present on LAMP carriers and bound to the WD40 domain of VPS41, enabling their recruitment. These findings reveal a unique role of VPS41 in recruiting TGN-derived LAMP carriers and expand our understanding of VPS41–Arl8b interactions beyond endosome–lysosome fusion, providing new insights into lysosomal trafficking mechanisms.


ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

February 2025

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22 Reads

The Ras homolog (Rho) small GTPases coordinate diverse cellular functions including cell morphology, adhesion and motility, cell cycle progression, survival, and apoptosis via their role in regulating the actin cytoskeleton. The upstream regulators for many of these functions are unknown. ARHGEF17 (also known as TEM4) is a Rho family guanine nucleotide exchange factor (GEF) implicated in cell migration, cell–cell junction formation, and the mitotic checkpoint. In this study, we characterize the regulation of the cell cycle by TEM4. We demonstrate that TEM4-depleted cells exhibit multiple defects in mitotic entry and duration, spindle morphology, and spindle orientation. In addition, TEM4 insufficiency leads to excessive cortical actin polymerization and cell rounding defects. Mechanistically, we demonstrate that TEM4-depleted cells delay in G1 as a consequence of decreased expression of the proproliferative transcriptional co-activator YAP. TEM4-depleted cells that progress through to mitosis do so with decreased levels of cyclin B as a result of attenuated expression of CCNB1. Importantly, cyclin B overexpression in TEM4-depleted cells largely rescues mitotic progression and chromosome segregation defects in anaphase. Our study thus illustrates the consequences of Rho signaling imbalance on cell cycle progression and identifies TEM4 as the first GEF governing Rho GTPase-mediated regulation of G1/S.


Sphingolipid metabolism orchestrates establishment of the hair follicle stem cell compartment

January 2025

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35 Reads

Sphingolipids serve as building blocks of membranes to ensure subcellular compartmentalization and facilitate intercellular communication. How cell type–specific lipid compositions are achieved and what is their functional significance in tissue morphogenesis and maintenance has remained unclear. Here, we identify a stem cell–specific role for ceramide synthase 4 (CerS4) in orchestrating fate decisions in skin epidermis. Deletion of CerS4 prevents the proper development of the adult hair follicle bulge stem cell (HFSC) compartment due to altered differentiation trajectories. Mechanistically, HFSC differentiation defects arise from an imbalance of key ceramides and their derivate sphingolipids, resulting in hyperactivation of noncanonical Wnt signaling. This impaired HFSC compartment establishment leads to disruption of hair follicle architecture and skin barrier function, ultimately triggering a T helper cell 2–dominated immune infiltration resembling human atopic dermatitis. This work uncovers a fundamental role for a cell state–specific sphingolipid profile in stem cell homeostasis and in maintaining an intact skin barrier.


FAM43A coordinates mtDNA replication and mitochondrial biogenesis in response to mtDNA depletion

Mitochondrial retrograde signaling (MRS) pathways relay the functional status of mitochondria to elicit homeostatic or adaptive changes in nuclear gene expression. Budding yeast have “intergenomic signaling” pathways that sense the amount of mitochondrial DNA (mtDNA) independently of oxidative phosphorylation (OXPHOS), the primary function of genes encoded by mtDNA. However, MRS pathways that sense the amount of mtDNA in mammalian cells remain poorly understood. We found that mtDNA-depleted IMR90 cells can sustain OXPHOS for a significant amount of time, providing a robust model system to interrogate human intergenomic signaling. We identified FAM43A, a largely uncharacterized protein, as a CHK2-dependent early responder to mtDNA depletion. Depletion of FAM43A activates a mitochondrial biogenesis program, resulting in an increase in mitochondrial mass and mtDNA copy number via CHK2-mediated upregulation of the p53R2 form of ribonucleotide reductase. We propose that FAM43A performs a checkpoint-like function to limit mitochondrial biogenesis and turnover under conditions of mtDNA depletion or replication stress.


TBC1D20 coordinates vesicle transport and actin remodeling to regulate ciliogenesis

TBC1D20 deficiency causes Warburg Micro Syndrome in humans, characterized by multiple eye abnormalities, severe intellectual disability, and abnormal sexual development, but the molecular mechanisms remain unknown. Here, we identify TBC1D20 as a novel Rab11 GTPase-activating protein that coordinates vesicle transport and actin remodeling to regulate ciliogenesis. Depletion of TBC1D20 promotes Rab11 vesicle accumulation and actin deconstruction around the centrosome, facilitating the initiation of ciliogenesis even in cycling cells. Further investigations reveal enhanced Rab11–MICAL1 interaction upon TBC1D20 loss, activating the monooxygenase domain of MICAL1 and inducing F-actin depolymerization around the centrosome. This actin network reorganization facilitates vesicle trafficking and docking, ultimately promoting ciliogenesis. In summary, our work uncovers a coordinated ciliogenesis initiation mechanism via Rab11 activation. These findings underscore a pivotal role for TBC1D20 in early ciliogenesis, advancing our understanding of its spatiotemporal regulation and offering insights into the disease pathogenesis associated with TBC1D20 mutations.


Translation of unspliced retroviral genomic RNA in the host cell is regulated in both space and time

January 2025

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13 Reads

Retroviruses carry a genomic intron-containing RNA with a long structured 5′-untranslated region, which acts either as a genome encapsidated in the viral progeny or as an mRNA encoding the key structural protein, Gag. We developed a single-molecule microscopy approach to simultaneously visualize the viral mRNA and the nascent Gag protein during translation directly in the cell. We found that a minority of the RNA molecules serve as mRNA and that they are translated in a fast and efficient process. Surprisingly, viral polysomes were also observed at the cell periphery, indicating that translation is regulated in both space and time. Virus translation near the plasma membrane may benefit from reduced competition for ribosomes with most cellular cytoplasmic mRNAs. In addition, local and efficient translation must spare energy to produce Gag proteins, where they accumulate to assemble new viral particles, potentially allowing the virus to evade the host’s antiviral defenses.


Arginylation of ⍺-tubulin at E77 regulates microtubule dynamics via MAP1S

Arginylation is the posttranslational addition of arginine to a protein by arginyltransferase-1 (ATE1). Previous studies have found that ATE1 targets multiple cytoskeletal proteins, and Ate1 deletion causes cytoskeletal defects, including reduced cell motility and adhesion. Some of these defects have been linked to actin arginylation, but the role of other arginylated cytoskeletal proteins has not been studied. Here, we characterize tubulin arginylation and its role in the microtubule cytoskeleton. We identify ATE1-dependent arginylation of ⍺-tubulin at E77. Ate1−/− cells and cells overexpressing non-arginylatable ⍺-tubulinE77A both show a reduced microtubule growth rate and increased microtubule stability. Additionally, they show an increase in the fraction of the stabilizing protein MAP1S associated with microtubules, suggesting that E77 arginylation directly regulates MAP1S binding. Knockdown of Map1s is sufficient to rescue microtubule growth rate and stability to wild-type levels. Together, these results demonstrate a new type of tubulin regulation by posttranslational arginylation, which modulates microtubule growth rate and stability through the microtubule-associated protein, MAP1S.


Rectification of planar orientation angle switches behavior and replenishes contractile junctions

January 2025

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5 Reads

In the early Drosophila embryo, germband elongation is driven by oriented cell intercalation through t1 transitions, where vertical (dorsal–ventral aligned) interfaces contract and then resolve into new horizontal (anterior–posterior aligned) interfaces. Here, we show that contractile events produce a continuous “rectification” of cell interfaces, in which interfaces systematically rotate toward more vertical orientations. As interfaces rotate, their behavior transitions from elongating to contractile regimes, indicating that the planar polarized identities of cell–cell interfaces are continuously re-interpreted in time depending on their orientation angle. Rotating interfaces acquire higher levels of Myosin II motor proteins as they become more vertical, while disruptions to the contractile molecular machinery reduce the rates of rotation. Through this angle rectification, the available pool of contractile interfaces is continuously replenished, as new interfaces acquire a contractile identity through rotation. Thus, individual cells acquire additional interfaces that are capable of undergoing t1 transitions, allowing cells to participate in multiple staggered rounds of intercalation events.


Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

January 2025

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20 Reads

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1 Citation

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4. We found that whereas CEP97 does not bind to microtubules directly, CP110 autonomously binds microtubule plus ends, blocks their growth, and inhibits depolymerization. Cryo-electron tomography revealed that CP110 associates with the luminal side of microtubule plus ends and suppresses protofilament flaring. CP110 directly interacts with CPAP, which acts as a microtubule polymerase that overcomes CP110-induced growth inhibition. Together, the two proteins impose extremely slow processive microtubule growth. Disruption of CP110–CPAP interaction in cells inhibits centriole elongation and increases incidence of centriole defects. Our findings reveal how two centriolar cap proteins with opposing activities regulate microtubule plus-end elongation and explain their antagonistic relationship during centriole formation.


Diverse microtubule-binding repeats regulate TPX2 activities at distinct locations within the spindle

TPX2 is an elongated molecule containing multiple α-helical repeats. It stabilizes microtubules (MTs), promotes MT nucleation, and is essential for spindle assembly. However, the molecular basis of how TPX2 performs these functions remains elusive. Here, we systematically characterized the MT-binding activities of all TPX2 modules individually and in combinations and investigated their respective contributions both in vitro and in cells. We show that TPX2 contains α-helical repeats with opposite preferences for “extended” and “compacted” tubulin dimer spacing, and their distinct combinations produce divergent outcomes, making TPX2 activity highly robust yet tunable. Importantly, a repeat group at the C terminus, R8-9, is the key determinant of the TPX2 function. It stabilizes MTs by promoting rescues in vitro and is critical in spindle assembly. We propose a model where TPX2 activities are spatially regulated via its diverse MT-binding repeats to accommodate its varied functions in distinct locations within the spindle. Furthermore, we reveal a synergy between TPX2 and HURP in stabilizing spindle MTs.


The KNL-1/Knl1 outer kinetochore protein caught regulating F-actin

January 2025

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9 Reads

Kinetochores are multiprotein complexes that link chromosomes to microtubules and are essential for chromosome segregation during cell divisions. In this issue, Alves Domingos et al. (https://doi.org/10.1083/jcb.202311147) show that the conserved KNL-1/Knl1 protein of the Knl1/Mis12/Ndc80 (KMN) outer kinetochore complex postmitotically regulates F-actin to shape somatosensory dendrites.


Classical cell cycle kinase limits tubulin polyglutamylation and cilium stability

January 2025

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5 Reads

Tubulin polyglutamylation is essential for maintaining cilium stability and function, and defective tubulin polyglutamylation is associated with ciliopathies. However, the regulatory mechanism underlying proper axonemal polyglutamylation remains unclear. He et al. (https://doi.org/10.1083/jcb.202405170) discovered that Cdk7/Cdk6/FIP5 phosphorylation cascade controls the ciliary import of tubulin glutamylases, thereby modulating axoneme polyglutamylation and ciliary signaling.


TanGIBLE: A selective probe for evaluating hydrophobicity-exposed defective proteins in live cells

The accumulation of defective polypeptides in cells is a major cause of various diseases. However, probing defective proteins is difficult because no currently available method can retrieve unstable defective translational products in a soluble state. To overcome this issue, there is a need for a molecular device specific to structurally defective polypeptides. In this study, we developed an artificial protein architecture comprising tandemly aligned BAG6 Domain I, a minimum substrate recognition platform responsible for protein quality control. This tandem-aligned entity shows enhanced affinity not only for model defective polypeptides but also for endogenous polyubiquitinated proteins, which are sensitive to translational inhibition. Mass-spectrometry analysis with this probe enabled the identification of endogenous defective proteins, including orphaned subunits derived from multiprotein complexes and misassembled transmembrane proteins. This probe is also useful for the real-time visualization of protein foci derived from defective polypeptides in stressed cells. Therefore, this “new molecular trap” is a versatile tool for evaluating currently “invisible” pools of defective polypeptides as tangible entities.


A STING–CASM–GABARAP pathway activates LRRK2 at lysosomes

January 2025

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66 Reads

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2 Citations

Mutations that increase LRRK2 kinase activity have been linked to Parkinson’s disease and Crohn’s disease. LRRK2 is also activated by lysosome damage. However, the endogenous cellular mechanisms that control LRRK2 kinase activity are not well understood. In this study, we identify signaling through stimulator of interferon genes (STING) as an activator of LRRK2 via the conjugation of ATG8 to single membranes (CASM) pathway. We furthermore establish that multiple chemical stimuli that perturb lysosomal homeostasis also converge on CASM to activate LRRK2. Although CASM results in the lipidation of multiple ATG8 protein family members, we establish that LRRK2 lysosome recruitment and kinase activation are highly dependent on interactions with the GABARAP member of this family. Collectively, these results define a pathway that integrates multiple stimuli at lysosomes to control the kinase activity of LRRK2. Aberrant activation of LRRK2 via this pathway may be of relevance in both Parkinson’s and Crohn’s diseases.


Differential impacts of ribosomal protein haploinsufficiency on mitochondrial function

The interplay between ribosomal protein (RP) composition and mitochondrial function is essential for energy homeostasis. Balanced RP production optimizes protein synthesis while minimizing energy costs, but its impact on mitochondrial functionality remains unclear. Here, we investigated haploinsufficiency for RP genes (rps-10, rpl-5, rpl-33, and rps-23) in Caenorhabditis elegans and corresponding reductions in human lymphoblast cells. Significant mitochondrial morphological differences, upregulation of glutathione transferases, and SKN-1–dependent oxidative stress resistance were observed across mutants. Loss of a Datasingle rps-10 copy reduced mitochondrial activity, energy levels, and oxygen consumption, mirrored by similar reductions in mitochondrial activity and energy levels in lymphoblast cells with 50% lower RPS10 transcripts. Both systems exhibited altered translation efficiency (TE) of mitochondrial electron transport chain components, suggesting a conserved mechanism to adjust mitochondrial protein synthesis under ribosomal stress. Finally, mitochondrial membrane and cytosolic RPs showed significant RNA and TE covariation in lymphoblastoid cells, highlighting the interplay between protein synthesis machinery and mitochondrial energy production.


Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

January 2025

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20 Reads

How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over 2 h. While a significant fraction of Condensins remains chromatin-bound until early G1, Cohesin-STAG1 and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes, and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in G1, is responsible for compaction inside TAD structures, and forms paired complexes upon completed nuclear import. Our quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by the sequential action of two Condensins in mitosis is seamlessly replaced by a less compact but conceptually similar hierarchically nested loop architecture driven by the sequential action of two Cohesins.



Journal metrics


7.8 (2022)

Journal Impact Factor™


7 days

Submission to first decision


2.1 (2022)

Immediacy Index


3.602 (2022)

Article Influence Score


$5,300 or $2,000

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