The IN/OUT assay: a new tool to study ciliogenesis

Kukic, Ira; Rivera‐Molina, Felix; Toomre, Derek

doi:10.1186/s13630-016-0044-2

Cited by 40 publications

(44 citation statements)

References 71 publications

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“…14). Cells treated with DiI-C6-TCO and HMSiR-Tz and imaged showed evidence of colocalization between DiI-HMSiR and the plasma membrane marker 40 sspH-mSmo 41 (Supplementary Fig. 15).…”

Section: Resultsmentioning

confidence: 99%

Long time-lapse nanoscopy with spontaneously blinking membrane probes

et al. 2017

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Long time-lapse, diffraction-unlimited super-resolution imaging of cellular structures and organelles in living cells is highly challenging, as it requires dense labeling, bright, highly photostable dyes, and non-toxic conditions. We developed a set of high-density, environment-sensitive (HIDE) membrane probes based on HMSiR that assemble in situ and enable long time-lapse, live cell nanoscopy of discrete cellular structures and organelles with high spatio-temporal resolution. HIDE-enabled nanoscopy movies are up to 50x longer than movies obtained with labeled proteins, reveal the 2D dynamics of the mitochondria, plasma membrane, and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum in living cells. These new HIDE probes also facilitate the acquisition of live cell, two-color, super-resolution images, greatly expanding the utility of nanoscopy to visualize processes and structures in living cells.

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“…14). Cells treated with DiI-C6-TCO and HMSiR-Tz and imaged showed evidence of colocalization between DiI-HMSiR and the plasma membrane marker 40 sspH-mSmo 41 (Supplementary Fig. 15).…”

Section: Resultsmentioning

confidence: 99%

Long time-lapse nanoscopy with spontaneously blinking membrane probes

et al. 2017

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“…To generate ecto-tagged β1 integrin pcDNA3 expression constructs, we used QuikChange Mutagenesis to insert a GAATTCCTCGAG sequence introducing unique EcoRI and XhoI sites into the ecto-domain coding region of pcDNA3 no-tag-β1 integrin between codons encoding Gly101 and Tyr102 (Supplementary Table 1 ). We PCR-amplified eGFP from the pEGFP-C2 vector (Clontech), ecliptic pHluorin from the sspH-mSmo vector 54 , HaloTag from C-Halo vector (Promega) and SNAP from pSNAP β Adrenergic Rc vector (New England BioLabs), using primers that added a 5′ linker containing an EcoRI site and a 3′ linker containing a XhoI site along with a spacer sequence (Supplementary Table 1 ). Each ecto-tag was then inserted into the β1 integrin ectodomain using the ectopic XhoI-EcoRI sites.…”

Section: Methodsmentioning

confidence: 99%

Novel ecto-tagged integrins reveal their trafficking in live cells

et al. 2017

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Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms. Integrin function is dynamically modulated by endo-exocytic trafficking, however, major mysteries remain about where, when, and how this occurs in living cells. To address this, here we report the generation of functional recombinant β1 integrins with traceable tags inserted in an extracellular loop. We demonstrate that these ‘ecto-tagged’ integrins are cell-surface expressed, localize to adhesions, exhibit normal integrin activation, and restore adhesion in β1 integrin knockout fibroblasts. Importantly, β1 integrins containing an extracellular pH-sensitive pHluorin tag allow direct visualization of integrin exocytosis in live cells and revealed targeted delivery of integrin vesicles to focal adhesions. Further, using β1 integrins containing a HaloTag in combination with membrane-permeant and -impermeant Halo dyes allows imaging of integrin endocytosis and recycling. Thus, ecto-tagged integrins provide novel powerful tools to characterize integrin function and trafficking.

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“…To highlight the advantages of the HIDE probe DiI‐SiR for STED nanoscopy of the plasma membrane, we compared the images obtained by visualizing DiI‐SiR to those obtained by visualizing SiR‐labeled Smo‐Halo, a widely used plasma membrane protein marker . HeLa cells expressing Smo‐Halo were treated with 2 μ m SiR‐CA for 30 min at 37 °C to form Smo‐SiR and visualized on the Leica SP8 STED microscope (Figure a and Figure S6).…”

Section: Figurementioning

confidence: 99%

Long‐Term Live‐Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI‐SiR

et al. 2017

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Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Here we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 sec temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 sec temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales. Keywordssuper-resolution microscopy; fluorophores; bioorthogonal chemistry; membranes; neurons Super-resolution microscopy (nanoscopy) techniques allow fluorescence microscopes to surpass the Abbe diffraction limit[1] and visualize structural features within cells with dimensions in the tens of nanometers.[2] But obtaining super-resolution images of live cells over long time periods (tens of minutes) remains a challenge[3] because: (1) the fluorophores in most samples are fused to proteins that are present at low density [4]; and (2) most fluorophores possess limited photostability and are inactivated within seconds. Fluorophore stability is a special concern for Stimulated Emission Depletion (STED) nanoscopy [5] because the sample is co-irradiated with an exceptionally powerful depletion laser.[2] We recently described a lipid-based strategy to visualize organelle structure and dynamics within live cells at super-resolution that is compatible with both STED [6] and Single Molecule Switching (SMS) [7] nanoscopy methods and overcomes both limitations. Prolonged, live-cell confocal and STED imaging of Golgi[6] structure and dynamics was Correspondence to: Derek K. Toomre; Alanna Schepartz. Here we extend the utility of this lipid-based strategy to achieve long time-lapse 2D imaging of the plasma membrane of cultured cells and primary neurons using STED nanoscopy. We find that the previously reported high-density lipid probe DiI-TCO, which localizes to the plasma membrane,[9] reacts readily with SiR-Tz[6] to form the HIDE probe DiI-SiR and enables live-cell STED imaging of HeLa cells ( Figure 1) for up to 25 min with 0.5 sec temporal resolution, corresponding to ~3000 time points, more than 70-fold longer than possible with Cer-SiR. HHS Public Access[6] We highlight the simplicity and versatility of imaging with DiISiR by observing the dynamics of day in vitro (DIV) 4 mouse hippocampal neurons for more than 9 min. To the best of our knowledge, these two movies represent the longest livecell STED movies of the plasma membrane in HeLa and primary neuronal cells with ≤ 2 sec temporal resolution. We anticipate that...

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The IN/OUT assay: a new tool to study ciliogenesis

Cited by 40 publications

References 71 publications

Long time-lapse nanoscopy with spontaneously blinking membrane probes

Long time-lapse nanoscopy with spontaneously blinking membrane probes

Novel ecto-tagged integrins reveal their trafficking in live cells

Long‐Term Live‐Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI‐SiR

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