Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: The biosynthetic LAMP1Δ-RUSH reporter system. (A) Schematic representation of the RUSH system. By co-expressing the ER hook streptavidin-KDEL with a reporter fused to SBP-GFP, the reporter can accumulate in the ER through the interaction between streptavidin and SBP. Addition of biotin allows for release of the reporter, which then traffics en masse through the secretory pathway. (B) Kinetic analysis of current RUSH cell line based on previous work. The type-1 membrane spanning RUSH reporter LAMP1Δ-GFP is used to monitor transport through the secretory system. Upon the addition of biotin, LAMP1Δ-RUSH traffics from the ER (0′), to the Golgi apparatus (15′), and then directly to the plasma membrane (30′–65′). From . Scale bar: 10 μm. (C) Lattice-SIM imaging allows the observation of LAMP1Δ-RUSH leaving the Golgi apparatus in tubular carriers (indicated by blue arrowheads) 35 min after biotin addition. In the example image (single micrograph from the time series), the cytosol can be seen full of these tubular structures. From . Scale bar: 10 μm; insert: 2 μm. (D) Time color-coded max projection of LAMP1Δ-RUSH carriers (represented by the RGB color bar 0–6s) shows their trajectory over time along the microtubular network (shown as a max projection). The insert shows a close-up example. From . Scale bar: 10 μm; insert: 2 μm. (E) Plasma membrane TIRF plane showing LAMP1Δ-RUSH post-Golgi tubule fusion. Tubules can be seen as bright spots as they approach and fuse, after which the cargo laterally diffuses on the plasma membrane. From . Scale bar: 10 μm; insert: 2 μm. (F) Schematic representation of the RUSH plus cell-surface staining protocol developed for flow cytometry analysis. (G) Quantitative cell-surface assay showing time course arrival of LAMP1Δ-RUSH to the plasma membrane after biotin addition. LAMP1Δ-RUSH cell line with a lumenal/extracellular GFP fusion was incubated with biotin for indicated times. Cells were subsequently labeled with a GFP binding nanobody fused to mCherry and underwent single-cell flow cytometry analysis. A minimum of 30,000 cells were analyzed for each biological repeat. The mean of the individual mCherry/GFP cell ratios were calculated for each experiment and are plotted on the graph (blue dots). Error bar = SD of at least three independent experimental repeats.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Expressing, Membrane, Imaging, Staining, Flow Cytometry, Incubation, Labeling, Binding Assay

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Lattice-SIM live-cell imaging of stable LAMP1Δ-RUSH HeLa cells showing the type-1 membrane spanning RUSH reporter LAMP1Δ-GFP trafficking from the ER (0′) to the Golgi apparatus (15′) and then directly to the plasma membrane (30′–65′). Cells were imaged every 30 s for 1 h after addition of biotin (500 μM). Scale bar: 10 μm. 10 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Membrane

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Lattice-SIM live-cell imaging of LAMP1Δ-RUSH leaving the Golgi apparatus of HeLa cells in tubular carriers 35 min after biotin addition (500 μM). Cells were imaged every 1.6 s for 5 min. Scale bar: 10 μm. 10 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Lattice-SIM live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with β-tubulin-mCherry (24 h). Video depicts LAMP1Δ-RUSH carriers (green) moving along the microtubular network (magenta). The cell was imaged 34 min after addition of biotin (500 μM) every 1.6 s for 2.4 min. Scale bar: 10 μm. 10 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: TIRF live-cell imaging of stable LAMP1Δ-RUSH HeLa cells 35 min after biotin addition (500 μM). Video shows the plasma membrane TIRF plane where LAMP1Δ-RUSH post-Golgi tubules can be seen fusing. Cells were imaged every 30 ms for 7 s. Scale bar: 10 μm. 50 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Membrane

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: LAMP1ΔYQTI-RUSH is glycosylated during RUSH assay. GFP immunoblot showing increasing LAMP1Δ-RUSH glycosylation over time after biotin addition. Note that LAMP1Δ-RUSH starts showing glycosylation from 20 min onwards. Gly = glycosylated LAMP1Δ-RUSH, Non-gly = non-glycosylated LAMP1Δ-RUSH.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: RAB6A, ARHGEF10, and RAB8A co-localize with LAMP1Δ-RUSH post-Golgi carriers. (A) Heterologous expression of HALO-RAB6A in WT HeLa cells shows RAB6A present at the Golgi and in tubular structures that bud off and travel toward the plasma membrane (single micrograph from a Lattice-SIM time series), as evidenced by blue arrowheads. From . Scale bar: 10 μm; insert: 2 μm. (B) Lattice-SIM imaging showing HALO-RAB6A (magenta) co-localizing with LAMP1Δ-RUSH tubules (green) leaving the Golgi and moving toward the plasma membrane (∼35 min after biotin addition). Orange arrowheads indicate co-localizing structures. From . Scale bar: 10 μm; insert: 2 μm. (C) Percent of LAMP1Δ-RUSH carriers positive for HALO-RAB6A, untagged HALO as a control, three biological repeats (total carriers quantified = 359), statistical analysis = two-tailed t test. (D) Lattice-SIM imaging showing co-localization of HALO-ARHGEF10 (magenta) with LAMP1Δ-RUSH carriers (green) traveling toward the plasma membrane (∼35 min after biotin addition). Orange arrowheads indicate co-localizing structures. From . Scale bar: 10 μm; insert: 2 μm. (E) Percent of LAMP1Δ-RUSH carriers positive for HALO-ARHGEF10, untagged HALO as a control, three biological repeats (total carriers quantified = 389), statistical analysis = two-tailed t test. (F) Plasma membrane TIRF plane showing HALO-RAB8A (magenta) co-localizing with LAMP1Δ-RUSH carriers (green) near their fusion site at the plasma membrane. The color white denotes co-localizing structures. From . Scale bar: 10 μm; insert: 2 μm. (G) Percent of LAMP1Δ-RUSH carriers positive for HALO-RAB8A, untagged HALO as a control, three biological repeats (total carriers quantified within 3 μm of plasma membrane edge = 285), statistical analysis = two-tailed t test. (H) Transient RAB6A KO dramatically reduces LAMP1Δ-RUSH at the plasma membrane in a quantitative cell-surface assay carried out 35 min after biotin addition. Note that expression of HALO - RAB6A WT (rescue), and constitutively active RAB6A (QL), is able to restore plasma membrane expression but not the constitutively inactive form (TN). (I) Widefield imaging of LAMP1Δ-RUSH WT and RAB6A transient KO, 1 h after biotin addition. Image shows the LAMP1Δ reporter unable to leave the Golgi in the RAB6A KO cells. Scale bar: 10 μm. (J) Widefield imaging of LAMP1Δ-RUSH reporter (green) in RAB6A KO cells expressing HALO-RAB6A (*rescue—magenta). Upon RAB6A heterologous expression, the reporter is at the plasma membrane 1 h after biotin addition. Scale bar: 10 μm. Nucleus stain = DAPI. Error bar = SD of at least three independent experimental repeats. Student’s t test was performed on data in C, E, and G, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in H. **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Expressing, Membrane, Imaging, Control, Two Tailed Test, Staining

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Lattice-SIM live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with HALO-RAB6A (24 h). Video shows HALO-RAB6A (magenta) co-localizing with LAMP1Δ-RUSH tubules (green) leaving the Golgi and moving toward the plasma membrane. The cell was imaged 21 min after biotin addition (500 μM), every 3.16 s for 4.2 min. Scale bar: 10 μm. 10 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection, Membrane

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Lattice-SIM live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with HALO-ARHGEF10 (24 h). Video shows co-localization of HALO-ARHGEF10 (magenta) with LAMP1Δ-RUSH carriers (green) traveling toward the plasma membrane. The cell was imaged 32 min after biotin addition (500 μM), every 3.16 s for 7.2 min. Scale bar: 10 μm. 10 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection, Membrane

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: TIRF live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with HALO-RAB8A (24 h). Video shows plasma membrane TIRF microscopy plane, where HALO-RAB8A (magenta) can be seen co-localizing with LAMP1Δ-RUSH carriers (green) near their fusion site. The cell was imaged 18 min after biotin every addition (500 μM), every 100 ms for 5.12 min. Scale bar: 10 μm. 50 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection, Membrane, Microscopy

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: ELKS and its associated proteins and/or homologs are not necessary for LAMP1Δ-RUSH post-Golgi tubule fusion. (A) TIRF imaging of LAMP1Δ-RUSH (gray) cells expressing heterologous HALO-ELKS (red) mostly localized to fusion sites (blue arrowheads). From . Scale bar: 10 μm. (B) Cell-surface ratio quantification (flow cytometry) of LAMP1Δ-RUSH at the plasma membrane after stable ELKS KO and 35 min of biotin exposure. (C) Immunoblot confirming loss of ELKS in a stable LAMP1Δ-RUSH ELKS KO clonal cell line. (D) Cell-surface ratio quantification (flow cytometry) of LAMP1Δ-RUSH at the plasma membrane after transient ELKS KO and 35 min of biotin exposure. (E) Cell-surface ratio quantification (flow cytometry) of LAMP1Δ-RUSH at the plasma membrane after transient KO of known ELKS interaction partners and 35 min of biotin exposure. This experiment was carried out in a stable clonal ELKS KO cell line. (F) Cell-surface ratio quantification (flow cytometry) of LAMP1Δ-RUSH at the plasma membrane after transient KO of EXOC1, ELKS, and combined. Error bar = SD of at least three independent experimental repeats. Two-tailed t test was performed on data in B and D, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in F. *P ≤ 0.05; ***P ≤ 0.001.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Imaging, Expressing, Flow Cytometry, Membrane, Western Blot, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: TIRF live-cell imaging of a EXOC3-KO HeLa cell stably expressing LAMP1Δ-RUSH and transfected with HALO-EXOC3 (24 h). Video shows co-localization of EXOC3-HALO (magenta) with LAMP1Δ-RUSH (green) carriers. The cell was imaged 17 min after biotin addition (500 μM), every 110 ms for 6 min. Scale bar: 10 μm. 50 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Locali z ation of exocyst subunits to LAMP1Δ-RUSH post-Golgi carriers, a C-terminal tag negatively affects EXOC6 function and validation of gene abrogation. (A) TIRF imaging of LAMP1Δ-RUSH (gray) cells expressing heterologous EXOC1-HALO (purple) after ∼35 min in biotin. From . Scale bar: 10 μm (B) TIRF imaging of LAMP1Δ-RUSH (gray) cells expressing heterologous HALO-EXOC6 (purple) after ∼35 min in biotin. From . Scale bar: 10 μm. (C) Immunoblot confirming loss of EXOC1 in a transient LAMP1Δ-RUSH EXOC1 KO cell population. (D) Recovery of EXOC6-HALO to EXOC6+6B KO cells, demonstrating a less efficient recovery than N-terminally tagged EXOC6 . (E) Overexpression of EXOC6-HALO has a moderate but significant effect on cell-surface delivery of LAMP1Δ-RUSH. (F) Validation of guide RNA KO efficiency by qRT-PCR. Data from all conditions was internally normalized to GAPDH expression and is represented as fold change of control LAMP1Δ-RUSH Cas9 cells. ELKS2/ERC2 and UNC13C were not detectable by qPCR in control conditions. Error bar = SD of at least three independent experimental repeats. Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in D and E. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Imaging, Expressing, Western Blot, Over Expression, Quantitative RT-PCR, Control

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: The exocyst complex subunits are essential for plasma membrane delivery. (A) Cell-surface ratio quantification (flow cytometry) assay on LAMP1Δ-RUSH at the plasma membrane after individual exocyst subunit KO and recovery with cDNA 35 min after biotin exposure. (B) Widefield imaging of LAMP1Δ-RUSH reporter in exocyst KO cells 1 h after biotin addition. When compared to WT, KO cells show substantial accumulation of post-Golgi LAMP1Δ-RUSH carriers. Scale bar: 20 μm; insert: 4 μm. Nucleus stain = DAPI. Error bar = SD of at least three independent experimental repeats. Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in A. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Membrane, Flow Cytometry, Imaging, Staining

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: Exocyst is recruited to post-Golgi carriers and multiple associated proteins are essential. (A) TIRF imaging of heterologous expression of EXOC3-HALO in EXOC3-KO LAMP1Δ-RUSH cells. EXOC3-HALO (magenta) specifically co-localizes with LAMP1Δ-RUSH (green) carriers near the plasma membrane. Orange arrowheads indicate co-localizing structures. From . Scale bar: 5 μm. (B) Percent of LAMP1Δ-RUSH carriers positive for EXOC3-HALO, untagged HALO as a control, three biological repeats (total carriers quantified within 3 μm of plasma membrane edge = 298), statistical analysis = two-tailed t test. (C) Percent of LAMP1Δ-RUSH carriers positive for EXOC1-HALO, untagged HALO as a control, three biological repeats (total carriers quantified within 3 μm of plasma membrane edge = 228), statistical analysis = two-tailed t test. (D) Percent of LAMP1Δ-RUSH carriers positive for HALO-EXOC6, untagged HALO as a control, three biological repeats (total carriers quantified within 3 μm of plasma membrane edge = 206), statistical analysis = two-tailed t test. (E) Schematic representation of RUSH carrierIP assay. (F) Gels containing resolved proteins from carrierIP assay denoting enrichment of exocyst subunits (HALO-tagged EXOC1, EXOC2, EXOC3, EXOC4, EXOC5, EXOC6, EXOC7, and EXOC8) in LAMP1Δ-RUSH post-Golgi carriers (LAMP1 immunoblot). Molecular weight markers are indicated in kD. (G) Cell-surface ratio quantification (flow cytometry) showing reduced amounts of LAMP1Δ-RUSH at the plasma membrane after transient KO of PIP5K homologs and 35 min of biotin exposure. (H) Widefield imaging of LAMP1Δ-RUSH reporter in triple PIP5K1A/B/C KO cells 1 h after biotin addition. When compared to WT, KO cells show accumulation of post-Golgi LAMP1Δ-RUSH carriers. Scale bar: 20 μm; insert: 4 μm. Nucleus stain = DAPI. Error bar = SD of at least three independent experimental repeats. Student’s t test was performed on data in B, C, and D, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in G. ***P ≤ 0.001.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Imaging, Expressing, Membrane, Control, Two Tailed Test, Western Blot, Molecular Weight, Flow Cytometry, Staining

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: TIRF live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with HALO-ELKS (24 h). Video shows LAMP1Δ-RUSH carriers (green) fusing in plasma membrane sites enriched for HALO-ELKS (magenta). Cells were imaged 31 min after biotin addition (500 μM), every 110 ms for 3.4 min. Scale bar: 10 μm. 100 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection, Membrane

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: TIRF live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with EXOC1-HALO (24 h). Video shows LAMP1Δ-RUSH carriers (green) co-localizing with EXOC1-HALO (magenta) near their fusion site. Cells were imaged 40 min after biotin addition (500 μM), every 50 ms for 3.15 min. Scale bar: 10 μm. 100 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: TIRF live-cell imaging of a HeLa cell stably expressing LAMP1Δ-RUSH and transfected with HALO-EXOC6 (24 h). Video shows LAMP1Δ-RUSH carriers (green) co-localizing with HALO-EXOC6 (magenta) near their fusion site. Cells were imaged 17 min after biotin addition (500 μM), every 110 ms for 6.2 min. Scale bar: 10 μm. 50 fps. See for stills.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection

Journal: The Journal of Cell Biology

Article Title: The exocyst complex is an essential component of the mammalian constitutive secretory pathway

doi: 10.1083/jcb.202205137

Figure Lengend Snippet: C- or N-terminally tagged LAMP1Δ-RUSH traffics with comparable kinetics to the cell surface. Widefield imaging series across different incubations of biotin (time indicated). Nucleus stain = DAPI. Scale bar: 20 μm. m, min.

Article Snippet: SBP-GFP-LAMP1ΔYQTI was PCR amplified from the original backbone SBP-GFP-LAMP1 ( ; a generous gift from Juan Bonifacino, National Institute of Child Health and Human Development, National Institutes of Health) and Gibson assembled (E2621L; New England Biolabs) to the pEGFP-C1 (Clontech) vector backbone in between the AgeI/HindIII restriction sites.

Techniques: Imaging, Staining

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Schematic representation of the YM201636 treatment and washout used for synchronization of endocytic lysosomal reformation (ELR) events. (B) Live-cell imaging of cells expressing LAMP1-GFP (green) and stained with LysoTracker Deep Red (magenta), following washout of YM201636 and recovery in drug-free media for 3 h. Images were acquired every 15 min to monitor the timeline and progression of ELR events. (C) Quantification of LAMP1-positive structures from (B). The line plot depicts the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; shaded regions or lines above and below represent the standard deviation (SD). (D) Quantification of LysoTracker-positive structures from (B). The line plot shows the number of LysoTracker-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; shaded regions or lines above and below represent the SD. (E) Super-resolution imaging of endolysosomes undergoing reformation using spinning disk confocal microscopy equipped with SoRa Disk. Cells expressing LAMP1-GFP (green) were treated with YM201636 for 2 h and imaged immediately after washout in drug-free media to capture ELR events. Images were acquired over 2 min at 2 sec intervals. White arrowheads indicate sites of fission and ELR events. (F) Quantification of data from (E). Table summarizing the percentage occurence of three distinct types of reformation events observed out of the first 100 events from 30 cells.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Live Cell Imaging, Expressing, Staining, Standard Deviation, Imaging, Confocal Microscopy

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell time-lapse imaging of cells expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta). Cells were treated with Nigericin to enlarge endocytic compartments. Nigericin was washed out and imaging was performed after 20 min of recovery. Each cell was imaged for 2 min at an interval of 2 sec. (B) Live-cell time-lapse imaging of cells expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta) treated with sucrose and invertase. Cells were fed with sucrose overnight and treated with invertase for 2 h before imaging to initiate ELR. Each cell was imaged for 2 min at an interval of 2 sec. (C) Immunofluorescent image of cells stained for LC3. Cells treated with YM20163 and recovery conditioned were fixed using 4%PFA while keeping starved and untreated cells as positive and negative control respectively. After fixation, coverslips were stained with anti-LC3 antibodies for imaging. (D) Immunoblot analysis to check the autophagic flux in cells treated with YM2016136 and recovery condition. Starvation with HBSS was used as a positive control for LC3 II lipidation. Tubulin was used as a loading control. (E) Fold change of LC3 II/LC3 I was assessed and plotted for bafilomycin positive conditions. Plot represents the mean fold change across n=3 biological replicates; One way ANOVA with Dunn’s multiple comparison; ***P= 0.0005 (untreated vs HBSS), ns P = 0.8087 (untreated vs 2 h YM201636), ns P = 0.2845 untreated vs 2 h YM201636 + 1h recovery)

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Imaging, Expressing, Staining, Negative Control, Western Blot, Positive Control, Control, Comparison

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot analysis of LAMP1 and Cathepsin D during reformation, using tubulin as a loading control. (B) Quantification of (A). Protein levels of LAMP1 and Cathepsin D at different maturation stages were analyzed for untreated, during YM201636 treatment, and post 1 h recovery. Fold change was determined by comparison to untreated control. Data represent mean of n=3 replicates; one-way ANOVA using Tukey’s multiple comparison, ns P = 0.8081 (Untreated vs 2 h YM201636), ns P= 0.4754 (Untreated vs 1 h recovery), ns P=0.2265 (2 h YM201636 vs 1 h recovery). For CTSD, ns P= 0.9871 (Untreated vs 2 h YM201636), ns P= 0.6745 (Untreated vs 1 h recovery), ns P=0.5877 (2 h YM201636 vs 1 h recovery) (C) Schematic representation of the analysis for cargo transport in tubules. (D) Super-resolution imaging of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (E) Intensity plot for (D), showing intensity values of green and magenta channels over distance (μm). (F) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Magic Red (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (G) Intensity plot for (F), showing intensity values of green and magenta channels over distance (μm). (H) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and Cathepsin D-RFP (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. (I) Intensity plot for (H), showing intensity values of green and magenta channels over distance (μm). (J) Super-resolution image of a tubulating endolysosome from a cell expressing LAMP1-GFP (green) and EGFR-mCherry (magenta) using a spinning disk confocal with SoRa disk. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate the ELR. Cells were also stimulated with EGF for 30 min to increase EGFR uptake. (K) Intensity plot for (J), showing intensity values of green and magenta channels over distance (μm).

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Western Blot, Control, Comparison, Imaging, Expressing, Staining

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell time-lapse imaging of cells expressing LAMP-GFP to assess the effect of translation inhibition on ELR kinetics. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate ELR. During recovery, cycloheximide was introduced in the recovery media while keeping media with ethanol as a vehicle control. Cells were imaged for 2 h at an interval of 15 min. (B) Quantification of LAMP1-positive structures from (A). The line plot shows the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; error bars represent the standard deviation (SD). C) Live-cell super resolution imaging of tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Lysotracker Deep Red (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding LysoTracker signal (magenta). (D) Live-cell super confocal imaging of tubulating endolysosome from a cell expressing LAMP1-GFP (green) and stained with Magic Red (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding Magic Red signal (magenta). E) Live-cell super resolution imaging of tubulating endolysosome from a cell co-expressing LAMP1-GFP (green) and EGFR-mCherry. Cells were stimulated with EGF for 30 min to increase EGFR uptake and imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1 positive compartments (green) and corresponding EGFR-mCherry signal (magenta). (F) Live-cell super confocal imaging of tubulating endolysosome from a cell co-expressing LAMP1-GFP (green) and cathepsin D-RFP (magenta). Cells were imaged for 2 min at an interval of 2 sec. Enlarged ROI shows the fission events of LAMP1-positive compartments (green) and corresponding cathepsin D signal (magenta).

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Imaging, Expressing, Inhibition, Control, Standard Deviation, Staining

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Western Blot, Phospho-proteomics, Positive Control, Inhibition, Control, Comparison, Live Cell Imaging, Expressing, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A ) Live-cell time-lapse imaging of cells expressing LAMP1–GFP to assess the effect of mTOR inhibition on ELR kinetics. Cells were treated with YM201636 for 2 h followed by washout and recovery to initiate ELR. During recovery, the mTOR inhibitor torin was added to the medium, with DMSO as vehicle control. Cells were imaged for 1 h at 15-min intervals. (B) Quantification of LAMP1-positive structures from (A). Line plot shows the number of LAMP1-positive compartments per cell over the recovery period. Data represent measurements from 30 cells. The central line indicates the mean; error bars indicate the standard deviation . ( C) Live-cell imaging of control knockout and dynamin-2 knockout cells expressing LAMP1-GFP to assess the role of dynamin 2 in endolysosomal tubule fission. Cells were imaged after 2 h YM201636 treatment followed by 30-min washout and recovery. Untreated cells were used as controls.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Imaging, Expressing, Inhibition, Control, Standard Deviation, Live Cell Imaging, Knock-Out

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell imaging of cells co transfected with LAMP1-GFP (green) and DRP1-mCherry (magenta) to assess the localization of DRP1 on lysosomes during reformation. Cells were imaged after treating with YM201636 for 2 h to inhibit the ELR and also after washout and 30 min of recovery to visualize the DRP1 on tubulating structures. (B) Co-localization of DRP1 with LAMP1 was measured in both YM201636 and recovery condition using Mander’s coefficient of DRP1 on LAMP1. Each dot represents the average Mander’s coefficient value of 100 μm 2 ROI. The plot shows mean Mander’s coefficient of 45 ROIs obtained from 15 cells in n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (YM201636 vs 30 min recovery). (C) Live-cell imaging of cells co transfected with LAMP1-GFP (green) and DRP1-mCherry (magenta) to assess the localization of DRP1 on lysosomes in untreated condition. (D) DRP1 (magenta) localized on the LAMP1-positive compartments (green) are highlighted in enlarged ROI using white arrowheads. The localization was also plotted using line plots measuring the gray values of LAMP1-GFP (green) and DRP1-mCherry (magenta) channels over the distance in µm. (E) Live-cell imaging of cells co-expressing LAMP-GFP and wild-type DRP1 or the DRP1 K38A mutant. (F) Quantification of (E). Number of tubular lysosomes were quantified and plotted. Each dot represents the number of LAMP1-positive tubules per 100 μm 2 ROI. The plot shows the mean number of tubular structures of 90 ROIs measure across 30 cells in n=3 replicate; Unpaired t -test: ****P = 0.4659 ×10 −6 (DRP1 WT vs DRP1 K38A). (G) Live-cell imaging of cells co-expressing LAMP-GFP and wild type DRP1 or the DRP1 K38A mutant. Cells were imaged after 2 h YM201636 treatment followed by washout and recovery of 30 min. (H) Quantification of LAMP1-positive tubule length from (G). Each data point represents the length of a single tubule. The plot shows the mean length of tubules from 30 cells across n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (DRP1 WT vs DRP1 K38A).

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Live Cell Imaging, Transfection, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Live-cell confocal microscopy of cells expressing LAMP1-GFP (green) and DRP1-mCherry treated with YM201636 for 2 h, followed by washout and recovery in drug-free imaging buffer. Images were captured every 2 sec. White arrows indicate a LAMP1-positive tubule undergoing fission and DRP1 recruitment at the fission site. (B) Intensity plot for (A), showing green and magenta channel intensities across distance (μm) at three time points during fission events. (C) Imaging of LAMP1-positive tubules in wild-type and DRP1 knockout (KO) cells, as well as DRP1 KO cells overexpressing wild-type DRP1 or the DRP1 K38A mutant. Cells were treated with YM201636 for 2 h, followed by 30 min washout and recovery. White arrows indicate LAMP1-positive tubules and correspond to DRP1 localization. (D) Quantification of LAMP1-positive tubule length from (C). Each data point represents the length of a single tubule. Data from n=3 replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P < 0.1×10 −14 (Control KO vs. DRP1 KO; DRP1 KO vs. DRP1 KO + WT DRP1; rescue with WT DRP1 vs. rescue with DRP1 K38A; Control KO vs. DRP1 KO + DRP1 K38A); ns, P > 0.9 ×10 −14 (Control KO vs. DRP1 KO + WT DRP1; DRP1 KO vs. DRP1 KO + DRP1 K38A) (E) Immunoblot analysis confirming absence of DRP1 in DRP1 KO cells and overexpression of DRP1 in rescue experiments. Tubulin was used as a loading control.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Confocal Microscopy, Expressing, Imaging, Knock-Out, Mutagenesis, Control, Western Blot, Over Expression

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Super-resolution imaging of endolysosomal tubules in cells expressing LAMP1-GFP (yellow), DRP1-mCherry (magenta), and stained with mitotracker deep red (cyan). Endolysosomal tubules were enriched using YM201636 and washout condition. (B) Intensity plot for data (A) obtained by plotting intensity values of LAMP1-GFP, DRP1-mCherry, and mitotracker channels over the distance in µm. (C) Super resolution imaging of endolysosomal tubules in cells expressing LAMP1-GFP (yellow), DRP1-mCherry (magenta), and KDEL-BFP (cyan). Endolysosomal tubules were enriched using YM201636 and washout condition. (D) Intensity plot for data (C) obtained by plotting intensity values of LAMP1-GFP, DRP1-mCherry, and KDEL-BFP over the distance in µm. (E) Super-resolution imaging of cells transfected with LAMP1-GFP (yellow), KDEL-BFP (magenta), and stained with mitotracker deep red (cyan). Endolysosomal tubules were enriched by YM201636 treatment for 2 h followed by washout and recovery in drug-free imaging buffer. White arrow indicates the contact site between ER, mitochondria, and endolysosomal tubule. (F) Intensity plots for data (E), showing intensities of all three channels along the line. Two intensity plots represent the proximity of three channels in two selected regions of interest (ROIs). (G) Live-cell super resolution microscopy shows contact sites exactly at the fission site. Cells were transfected with LAMP1-GFP (yellow), KDEL-BFP (magenta), and stained with mitotracker deep red (cyan). Fission events were enriched using YM201636 treatment for 2 h followed by washout and recovery. Cells were recorded for 2 min at 2 second intervals. The white arrowhead indicates the fission site. (H) Quantification of endolysosomal tubule fission events based on proximity to ER, mitochondria, both, or none. 100 fission events were counted from n=3 biological replicates.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Imaging, Expressing, Staining, Transfection, Super-Resolution Microscopy

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Disruption of ER contact by overexpressing CLIMP63. Cells were transfected with LAMP1-GFP (green) and KDEL-mCherry or CLIMP63-mCherry. Imaging was performed after 2 h of YM201636 treatment, washout, and 30 min of recovery. White arrows depict the LAMP1-positive compartment with respective ER morphology. (B) Schematic representation of CLIMP63 overexpression and its implication on ELR. (C) Mitochondrial morphology was disturbed by treating cells with 20 μM CCCP for 30 min. Cells expressing LAMP1-GFP (green) were treated with YM201636 for two h followed by washout and 30 min recovery. Twenty μM CCCP was introduced in the recovery media. Mitochondria were stained using MitoTracker Deep Red (magenta). Images were taken after 30 min of recovery to observe the effect. White arrow shows the morphology of the LAMP1-positive compartment in DMSO versus CCCP-treated cells. (D) Schematic representation of CCCP treatment on mitochondria and its implication on ELR. (E) Live-cell imaging of LAMP1-positive lysosomes (green) in control KO and MFF knockout (KO) cells. Cells were imaged after staining with MitoTracker Deep Red (magenta). White arrowheads highlight the LAMP-1positive compartments in the ROIs. (F) Quantification of tubules length of endolysosomes from data (A). Each dot represents the length of individual tubules and the plot shows the mean of 90 tubules measured in 30 cells across n=3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (KDEL OE vs CLIMP OE). (G) Quantification of the average area of LAMP1-positive structures for (C). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n= 3 biological replicates; Unpaired t -test: ****P < 0.1 ×10 −14 (DMSO vs CCCP). (H) Quantification of the average area of LAMP1-positive structures for (E). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biolog ical replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P = 0.5356 ×10 −4 (Control KO vs MFF KO C1), ****P = 0.2273 ×10 −4 (Control KO vs MFF KO C 2), ns P > 0.9 ×10 −14 (MFF KO C1 vs MFF KO C2).

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Disruption, Transfection, Imaging, Over Expression, Expressing, Staining, Live Cell Imaging, Control, Knock-Out

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Schematic representation of calcium transport during ELR. (B) Schematic representation of the assay used to inhibit the lysosomal calcium exporter (TRPML1) or the mitochondrial calcium importer (VDAC) to assess its effect on ELR. (C) Live-cell imaging of cells co-transfected with LAMP1-GFP (green) and the mitochondrial calcium sensor mt-RCAMP1h (magenta). ELR was initiated with 2 h YM201636 treatment and washout. Cells were imaged for 30 min at 15-min intervals. D) Mean fluorescence intensity of mt-RCAMP1h (magenta) was quantified and plotted for 0 min, 15 min and 30 min recovery timepoints. Each dot represents the gray value of an individual cell. The plot shows the mean fluorescence intensity of 30 cells from n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ****P = 0.5257 × 10 −4 (YM201636 vs. 15 min recovery), ****P = 0.03522 × 10 −6 (YM201636 vs. 30 min recovery), ns P > 0.9473 (15 min recovery vs. 30 min recovery). E) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the TRPML1 inhibitor ML-SI3. Cells were subsequently washed and recovered in media containing only ML-SI3 to test the effect of ML-SI3 alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (F) Quantification of the average area of LAMP1-positive structures for (E). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with ML-SI3), ****P = 0.691 × 10 −12 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 15 min recovery), ns P > 0.9 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and ML-SI3 + 30 min recovery), ****P = 0.4885 ×10 −4 (2 h YM201636 + 30 min recovery with ML-SI3 vs. 2 h YM201636 and ML-SI3 + 15 min recovery), ****P = < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery with ML-SI3 vs. 2 h YM201636 and ML-SI3 + 30 min recovery). (G) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the VDAC2/3 inhibitor erastin. Cells were subsequently washed and recovered in media containing only erastin to test the effect of erastin alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (H) Quantification of the average area of LAMP1-positive structures for (G). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with Erastin), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 15 min recovery), ns P > 0.9 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and Erastin + 30 min recovery), **P = 0.00103 (2 h YM201636 + 30 min recovery with Erastin vs. 2 h YM201636 and Erastin + 15 min recovery), ****P = < 0.9 ×10 −14 (2 h YM201636 + 30 min recovery with Erastin vs. 2 h YM201636 and Erastin + 30 min recovery). (I) Live-cell imaging of cells expressing LAMP1-GFP and treated with YM201636 together with the VDAC1 inhibitor VBIT-4. Cells were subsequently washed and recovered in media containing only VBIT-4 to test the effect of VBIT-4 alone, as described in (B). ROIs show the LAMP1-positive compartments in representative parts of cells. (J) Quantification of the average area of LAMP1-positive structures for (I). Each dot represents the average area per 100 µm² ROI. The plot shows the mean area from a total of 90 ROIs from 30 cells across n = 3 biological replicates; Kruskal-Wallis test with Dunn’s multiple comparisons: ns P > 0.9 ×10 −14 ( 2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 30 min recovery), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 + 30 min recovery with VBIT-4), ****P < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 15 min recovery), ns P =0.2364 (2 h YM201636 + 30 min recovery vs. 2 h YM201636 and VBIT-4 + 30 min recovery), **P = 0.29 ×10 −4 (2 h YM201636 + 30 min recovery with VBIT-4 vs. 2 h YM201636 and VBIT-4 + 15 min recovery), ****P = < 0.1 ×10 −14 (2 h YM201636 + 30 min recovery with VBIT-4 vs. 2 h YM201636 and VBIT-4 + 30 min recovery).

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Live Cell Imaging, Transfection, Fluorescence, Expressing

Journal: Cell reports

Article Title: The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.

doi: 10.1016/j.celrep.2021.109945

Figure Lengend Snippet: Figure 1. Rab5 and Rab7 are mostly absent from CPP-containing endosomes (A–D) HeLa KCNN4 knockout cells ectopically expressing early (Rab5A, Rab5B and EEA1) or late (Rab7 and Lamp1) endosomal markers were incubated with 20 mg/ml AlexaFluor568-transferrin, 0.2 mg/ml TMR-10 kDa dextran, 2 mg/ml AlexaFluor647-EGF or with 40 mM of TMR- (TAT, R9, Penetratin, TAT-RasGAP317-

Article Snippet: GFP-hEEA1 (#970) and hLamp1-GFP.dn3 (#971) encoding GFP-labeled versions of human EEA1 and Lamp1, respectively were from Addgene (Cat# 42307 and 34831).

Techniques: Knock-Out, Expressing, Incubation

Journal: Cell reports

Article Title: The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.

doi: 10.1016/j.celrep.2021.109945

Figure Lengend Snippet: Figure 2. CPP endocytosis does not require PI(3)P-kinase-like enzymes (A) Colocalization quantitation between the indicated endosomal material and endosomal markers in the presence or in the absence of LY294002, a pan-PI3- kinase inhibitor. HeLa KCNN4 knockout cells, ectopically expressing GFP-tagged EEA1 or Lamp1, were incubated or not for 30 min with 25 mM LY294002 in RPMI without serum. The cells were then incubated for a 5-minute pulse in RPMI, 10% FBS with either 20 mg/ml AlexaFluor568-transferrin, 0.2 mg/ml TMR-10 kDa dextran or 40 mM TMR-R9, washed, and then visualized by confocal microscopy over time (time 0 corresponds to the addition of the cargos). When used, LY294002 was maintained in the media throughout the full duration of the experiment. The data correspond to the mean ± SD of 160 cells (per condition) sampled from three independent experiments. The p values were calculated on area under the curve (AUC) values using parametric unpaired two-tailed t tests. (B) Quantitation of the number of endosomal vesicles per cell in the presence or in the absence of wortmannin, a pan-PI3-kinase inhibitor. HeLa KCNN4 knockout cells were incubated with 20 mg/ml AlexaFluor568-transferrin, 0.2 mg/ml TMR-10 kDa dextran or 40 mM TMR-CPP for a pulse of 5 min. Cells were preincubated or not for 30 min with 10 mM wortmannin, which was still present during the full duration of the experiment. The number of vesicles positive for the indicated en- dosomal material were visually counted from confocal images, acquired in the middle of the cell. The results are derived from the analysis of 160 cells (per condition) taken from three independent experiments. The p values were calculated using parametric paired two-tailed t test. See also Figure S4.

Article Snippet: GFP-hEEA1 (#970) and hLamp1-GFP.dn3 (#971) encoding GFP-labeled versions of human EEA1 and Lamp1, respectively were from Addgene (Cat# 42307 and 34831).

Techniques: Quantitation Assay, Knock-Out, Expressing, Incubation, Confocal Microscopy, Two Tailed Test, Derivative Assay

Journal: Cell reports

Article Title: The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.

doi: 10.1016/j.celrep.2021.109945

Figure Lengend Snippet: Figure 3. CPP endosomal maturation is Rab14-dependent (A) Colocalization quantitation between TMR-TAT and GFP-EEA1 (15 min) or Lamp1-GFP (30 min) in a pulse-chase experiment performed in MDCK-II wild-type cells and the indicated Rab knockouts. A total of 50–65 cells were quantitated per condition. Statistical analysis was performed with ANOVA multiple comparison to the wild-type cell condition with Dunett’s correction. Only significant p values are shown. Rab5-degron cells were treated with 1 mg/ml doxycycline and 500 mM IAA for 48 h to induce degradation of degron-tagged Rab5A in the Rab5B/C knockout cell line (see Figure S5A). (B) Rab14 rescue experiment. Rab14 knockout cells were transiently transfected (KO + Rab14) or not (KO) with a GFP-Rab14 wild-type construct. These cells, along wild-type MDCK cells (WT), were then analyzed as in (A) (55–58 cells per con- dition were quantitated). See also Figures S5 and S6.

Article Snippet: GFP-hEEA1 (#970) and hLamp1-GFP.dn3 (#971) encoding GFP-labeled versions of human EEA1 and Lamp1, respectively were from Addgene (Cat# 42307 and 34831).

Techniques: Quantitation Assay, Pulse Chase, Comparison, Knock-Out, Transfection, Construct

Journal: Cell reports

Article Title: The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.

doi: 10.1016/j.celrep.2021.109945

Figure Lengend Snippet: Figure 5. HDs are following a Rab5-independent, Rab14-dependent endocytic route (A and B) Colocalization quantitation between endosomal markers and HD. Wild-type (A) and KCNN4 knockout (B) HeLa cells, ectopically expressing the indicated endosomal markers, were subjected to a 5-minute pulse incubation with fluorescently labeled OTX2 HD (10 mM). Quantitation assessment was based on confocal images. The results correspond to the mean ± SD of 150–170 analyzed cells per condition derived from three independent experiments. (C) Representative confocal images of the indicated MDCK-II cell lines expressing BFP-EEA1 incubated with 10 mM FITC-OTX2 for a 5-min pulse. The images correspond to the 15 min time point. Scale bar: 10 mm. (D) Colocalization quantitation between fluorescent versions of OTX2 HD (10 mM) with EEA1 (top) or Lamp1 (bottom) in wild-type and the indicated Rab knockout MDCK-II cells. Cells were incubated for 5 min, then washed and confocal images were acquired at 15 and 30 min post incubation for early and late endosomal markers, respectively. A total of 54–60 cells per condition were analyzed. Statistical analysis was performed with ANOVA multiple comparison to wild-type condition with Dunett’s correction. (E) Quantitation of total OTX2 HD uptake in MDCK-II wild-type and Rab14 knockout cells. Statistical analysis was done with parametric unpaired t test. A total of 130 cells per condition were analyzed.

Article Snippet: GFP-hEEA1 (#970) and hLamp1-GFP.dn3 (#971) encoding GFP-labeled versions of human EEA1 and Lamp1, respectively were from Addgene (Cat# 42307 and 34831).

Techniques: Quantitation Assay, Knock-Out, Expressing, Incubation, Labeling, Derivative Assay, Comparison

Journal: Cell reports

Article Title: The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.

doi: 10.1016/j.celrep.2021.109945

Figure Lengend Snippet: Figure 6. Polyamines are following a Rab5-independent, Rab14-dependent endocytic route (A and B) Colocalization quantitation between endosomal markers and polyamine. Wild-type (A) and KCNN4 knockout (B) HeLa cells, ectopically expressing RFP-Rab5A, BFP-EEA1, mCherry-Rab7, BFP-LAMP1, or RFP-Rab14, were subjected to a 5 min pulse with fluorescently labeled spermine (5 mM). Quantitation assessment was based on confocal images. The results correspond to the mean ± SD of 150–170 analyzed cells per condition derived from three independent experiments. (C) Representative confocal images of the indicated MDCK-II cell lines ectopically expressing BFP-EEA1, incubated with 5 mM CF488-spermine for a 5-min pulse. The images correspond to the 15 min time point condition. Scale bar: 10 mm. (D) Colocalization quantitation between fluorescent versions of spermine (5 mM) with BFP-EEA1 (top) or BFP-Lamp1 (bottom) in wild-type and the indicated Rab knockout MDCK-II cells. Cells were incubated for 5 min, then washed and confocal images were acquired at 15 and 30 min post incubation for early and late endosomal markers, respectively. A total of 54–60 cells per condition were analyzed. Statistical analysis was performed with ANOVA multiple comparison to the wild-type condition with Dunett’s correction. (E) Wild-type and Rab14 knockout MDCK-II cells were incubated with 5 mM CF488-labeled spermine for a 5-min pulse and then analyzed 10 min later. Statistical analysis was done with parametric unpaired t test. A total of 130 cells per condition were analyzed. (legend continued on next page)

Article Snippet: GFP-hEEA1 (#970) and hLamp1-GFP.dn3 (#971) encoding GFP-labeled versions of human EEA1 and Lamp1, respectively were from Addgene (Cat# 42307 and 34831).

Techniques: Quantitation Assay, Knock-Out, Expressing, Labeling, Derivative Assay, Incubation, Comparison

Journal: Cell reports

Article Title: The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7.

doi: 10.1016/j.celrep.2021.109945

Figure Lengend Snippet: Figure 7. Acidic nature of the CPP- and Lamp1-positive endosomes (A) Representative confocal images of wild-type HeLa cells expressing Lamp1-BFP incubated for one hour with 1 mM LysoSensor Green DND-189. Scale bar: 10 mm. (B) Colocalization quantitation of acidic Lamp1 vesicles based on confocal images in wild-type and KCNN4 KO HeLa cells incubated with the indicated cargos for one hour (58 cells per condition were analyzed). Statistical analysis was performed using ANOVA test with Dunett’s correction. The p values correspond to the comparison with dextran. In this graph, all Lamp1-positive vesicles are considered, not only those that colocalize with the indicated markers. (C) Representative confocal images of KCNN4 knockout HeLa cells expressing Lamp1-BFP incubated for one hour with 40 mM TMR-TAT and 1 mM LysoSensor Green DND-189. Scale bar: 10 mm. (D) Colocalization quantitation of the indicated endocytic material with lysosensor in wild-type HeLa cells based on visual quantitation (left) or Mander’s coefficient (70 cells per condition were analyzed). The cargos (40 mM TMR-TAT, 10 mM OTX2 HD, 5 mM CF647-labeled spermine, or 0.2 mg/ml TMR-10 kDa dextran) and lysosensor (1 mM) were incubated with the cells for one hour. Statistical analysis was performed using ANOVA test with Dunett’s correction. The p values correspond to the comparison with cells incubated with dextran. See also Figure S7.

Article Snippet: GFP-hEEA1 (#970) and hLamp1-GFP.dn3 (#971) encoding GFP-labeled versions of human EEA1 and Lamp1, respectively were from Addgene (Cat# 42307 and 34831).

Techniques: Expressing, Incubation, Quantitation Assay, Comparison, Knock-Out, Labeling

Journal: bioRxiv

Article Title: A genome wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity

doi: 10.1101/2025.02.28.640639

Figure Lengend Snippet: (A) Mouse plasma cell subsets from the spleen and bone marrow were sorted and stained for surface CD138 (pink) and intracellular ATP6V1A protein (cyan) and cells analyzed by imaging flow cytometry. ATP6V1A spot numbers/cell were enumerated and mean +/- SEM shown. Combined data from 9890-10755 cells from 12 mice across 3 experiments. *p<0.05 by Games-Howell’s multiple comparisons test. (B) Mouse plasma cell subsets as in (A) were stained for surface CD138 (pink) and RAB7 (cyan). RAB7+ spot numbers/cell were enumerated and mean +/- SEM graphed. Pooled data from 1639-2115 cells from 12 mice across 3 experiments. *p<0.05 by Games-Howell’s multiple comparisons test. (C) Mouse plasma cell subsets as in (A) were stained for surface CD138 (pink) and LAMP1 (cyan). LAMP1+ spot numbers/cell were enumerated and mean +/- SEM graphed. Pooled data from 3061-9159 cells from 12 mice across 3 experiments. *p<0.05 by Games-Howell’s multiple comparisons test. (D) Human plasma cells from the tonsil (CD19+ CD38+ CD27+ CD138-) and bone marrow CD19+ and CD19- plasma cells (CD27+ CD38+ CD138+) were stained for surface CD38 (orange) and intracellular ATP6V1A protein. ATP6V1A+ spot numbers/cell were quantified and mean +/- SEM plotted for all subsets. Combined data from 1156-9050 cells from 7 tonsil donors and 6 bone marrow donors across 3 experiments. *p<0.05 by Games-Howell’s multiple comparisons test. (E) Human plasma cell subsets as in (D) were stained for surface CD38 and intracellular LAMP1. LAMP1+ spot numbers/cell were quantified and mean +/- SEM plotted. Pooled data from 2614-10742 cells from 9 tonsil donors and 7 bone marrow donors from 4 experiments. *p<0.05 by Games-Howell’s multiple comparisons test.

Article Snippet: The mouse Brie CRISPR-knockout library (73633), lentiGuide-puro (52963), pLenti- sfGFP-LAMP1-mCherry (164478), and pHAGE-DDX3X (116730) were all purchased from Addgene ( , , , ).

Techniques: Staining, Imaging, Flow Cytometry

Journal: bioRxiv

Article Title: A genome wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity

doi: 10.1101/2025.02.28.640639

Figure Lengend Snippet: (A) 5TGM1-Cas9 cells transduced with control gRNA targeting the Rosa26 locus (black) and Pi4kb -targeting gRNA (yellow) and then scored for intracellular ATP6V1A+ spots/cell. Representative images for each of the groups (left) and numbers of ATP6V1A+ spots/cell (right) were quantified. Pooled data from 4236-4368 cells across 3 independent experiments. *p<0.05 by an unpaired t-test with Welch’s correction. (B) Groups in (A) were scored for intracellular LAMP1+ spots/cell. Representative images for each of the groups (left) and numbers of LAMP1+ spots/cell (right) were quantified. Pooled data from 4830-4837 cells across 3 independent experiments. *p<0.05 by an unpaired t-test with Welch’s correction. (C) 5TGM1-Cas9-pHLARE cells were transduced with lentivirus containing gRNA targeting Pi4kb along with a control gRNA. Representative histogram (left) of the ratio of sfGFP to mCherry MFI in control gRNA (black) and Pi4kb -targeting gRNA (yellow) groups. Quantification of mean sfGFP/mCherry ratio (right) in control and knockout cultures. Each circle represents a single group and groups within the same experiment are joined by a line. Pooled data from 7 independent experiments. *p<0.05 by a paired t-test. (D) Surface CD98 expression on groups in (C). Representative histogram (left) and quantified CD98 MFI (right) are shown. Combined data from 13 experiments. *p<0.05 by a paired t-test. (E) Secreted IgG2b from culture supernatant of groups in (C) were quantified by ELISA. AUC for each group is shown. Pooled data from 7 experiments. *p<0.05 by a paired t- test.

Article Snippet: The mouse Brie CRISPR-knockout library (73633), lentiGuide-puro (52963), pLenti- sfGFP-LAMP1-mCherry (164478), and pHAGE-DDX3X (116730) were all purchased from Addgene ( , , , ).

Techniques: Transduction, Control, Knock-Out, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1

doi: 10.1038/s41419-018-1056-1

Figure Lengend Snippet: Neimann–Pick disease type C1-specific proteins that are differentially expressed by treatment with HPβCD or HPγCD. LAMP-1 protein was identified as significantly upregulated upon HPγCD treatment (also upon HPβCD treatment when compared with the NPC group) as compared to either healthy or NPC control cells

Article Snippet: LAMP-1 expression construct pCMV6-AC-GFP-LAMP-1 (RG219208), LAMP-1 knock down plasmid pGFP-C-shLenti (TL311795), and scrambled shRNA plasmid (TR30021) were purchased from Origene (Rockville, MD, USA).

Techniques: Control, Membrane