A PDMS-based biochip with integrated sub-micrometre position control for TIRF microscopy of the apical cell membrane

Thuenauer, Roland; Juhász, Kata; Mayr, Reinhard; Frühwirth, Thomas; Lipp, Anna-Maria; Balogi, Zsolt; Sonnleitner, Alois

doi:10.1039/c1lc20458k

Cited by 18 publications

(20 citation statements)

References 42 publications

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“…The chip allows approaching the cells to the glass surface with submicrometer precision so that the apical PM can be imaged using selective excitation via a shallow (∼200 nm) evanescent wave (32). This technique allows observation of individual apical vesicle fusion events, including the subsequent diffusive spreading of cargo proteins (17). Indeed, starting at ∼1 h after release from the ER, we observed vesicles bearing rhodopsin-GFP fusing with the apical PM.…”

Section: Rab11a Is Present On Rhodopsin Vesicles Undergoing Apical Mementioning

confidence: 97%

“…The procedure for apical TIRFM by means of a biochip has been described previously (17). Briefly, MDCK II cells were grown on a fibronectin-coated cell culture area on top of the biochip for 4 d before transfection with the indicated plasmids.…”

Section: Methodsmentioning

confidence: 99%

“…To image the progression of rhodopsin-GFP from Rab11a-positive AREs to the apical PM, we used a recently developed biochip that enables TIRFM at the apical PM of polarized epithelial cells (17). Briefly, after a polarized monolayer of MDCK cells is grown on the biochip, the whole chip is inverted and placed on a glass coverslip on a microscope.…”

Section: Rab11a Is Present On Rhodopsin Vesicles Undergoing Apical Mementioning

confidence: 99%

“…Here, we report experiments in which we studied the biosynthetic trans-endosomal trafficking of an apical reporter, rhodopsin-GFP (14,15), synchronized by reversible aggregation at the endoplasmic reticulum (ER) through recombinant tagging with conditional aggregation domains (CADs) (16). Post-Golgi trafficking through endosomal compartments of rhodopsin-GFP, freed from the CADs through a furin-cleavage site, was traced and quantified by spinning disk confocal microscopy and its surface delivery was analyzed using a unique total internal reflection fluorescence microscopy (TIRFM) protocol that allows live imaging of the apical PM (17).…”

Section: Significancementioning

confidence: 99%

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Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates

Thuenauer

Hsu

Carvajal-González

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane. P olarized epithelial cells maintain separate apical and basolateral plasma membrane (PM) domains to carry out essential vectorial absorptive and secretory functions. Tightly regulated vesicular trafficking is required to sort and target distinct sets of proteins to their respective membranes (1). Studies performed with the epithelial model cell line Madin-Darby canine kidney (MDCK) indicate that newly synthesized membrane proteins take an indirect route to the PM. After leaving the trans-Golgi network (TGN), PM proteins first traverse endosomal compartments before reaching apical or basolateral cell surfaces (2-4). Some basolateral PM proteins traverse common recycling endosomes (CREs) where they are sorted into carrier vesicles directed toward the basolateral PM by clathrin and the clathrin adaptor AP-1B (5, 6). Alternatively, basolateral proteins can be sorted at the TGN by the clathrin adaptor AP-1A (7-9). Along the apical route it has been shown that nonraft-associated apical proteins traverse Rab11-positive apical recycling endosomes (AREs) en route to the apical PM (10). Trans-endosomal trafficking is likely to play important roles in cell physiology; for example, endosomal compartments may function ...

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Section: Rab11a Is Present On Rhodopsin Vesicles Undergoing Apical Mementioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Rab11a Is Present On Rhodopsin Vesicles Undergoing Apical Mementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates

Thuenauer

Hsu

Carvajal-González

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…10). First, ascribing to microfluidic devices enabling graded control of respective environmental cues [45][46][47][48] (Figs. 6(b), 7(a), and 7(b)), integration of multi-level, multi-factor controls in the system could elicit combinations of system inputs in FSC strategy, which is aimed to search optimized combinations of environmental cues that support long-term maintenance of specific morphological variants (goal 1 in Fig.…”

Section: A Practical Research Strategymentioning

confidence: 99%

Morphological plasticity of bacteria—Open questions

Shen

Chou

2016

Biomicrofluidics

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Morphological plasticity of bacteria is a cryptic phenomenon, by which bacteria acquire adaptive benefits for coping with changing environments. Some environmental cues were identified to induce morphological plasticity, but the underlying molecular mechanisms remain largely unknown. Physical and chemical factors causing morphological changes in bacteria have been investigated and mostly associated with potential pathways linked to the cell wall synthetic machinery. These include starvation, oxidative stresses, predation effectors, antimicrobial agents, temperature stresses, osmotic shock, and mechanical constraints. In an extreme scenario of morphological plasticity, bacteria can be induced to be shapeshifters when the cell walls are defective or deficient. They follow distinct developmental pathways and transform into assorted morphological variants, and most of them would eventually revert to typical cell morphology. It is suggested that phenotypic heterogeneity might play a functional role in the development of morphological diversity and/or plasticity within an isogenic population. Accordingly, phenotypic heterogeneity and inherited morphological plasticity are found to be survival strategies adopted by bacteria in response to environmental stresses. Here, microfluidic and nanofabrication technology is considered to provide versatile solutions to induce morphological plasticity, sort and isolate morphological variants, and perform single-cell analysis including transcriptional and epigenetic profiling. Questions such as how morphogenesis network is modulated or rewired (if epigenetic controls of cell morphogenesis apply) to induce bacterial morphological plasticity could be resolved with the aid of micro-nanofluidic platforms and optimization algorithms, such as feedback system control.

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Nanostructured‐Based Optical Readouts Interfaced with Machine Learning for Identification of Extracellular Vesicles

Mata

Jeanne

Jalali

et al. 2022

Adv Healthcare Materials

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Extracellular vesicles (EVs) are shed from cancer cells into body fluids, enclosing molecular information about the underlying disease with the potential for being the target cancer biomarker in emerging diagnosis approaches such as liquid biopsy. Still, the study of EVs presents major challenges due to their heterogeneity, complexity, and scarcity. Recently, liquid biopsy platforms have allowed the study of tumor‐derived materials, holding great promise for early‐stage diagnosis and monitoring of cancer when interfaced with novel adaptations of optical readouts and advanced machine learning analysis. Here, recent advances in labeled and label‐free optical techniques such as fluorescence, plasmonic, and chromogenic‐based systems interfaced with nanostructured sensors like nanoparticles, nanoholes, and nanowires, and diverse machine learning analyses are reviewed. The adaptability of the different optical methods discussed is compared and insights are provided into prospective avenues for the translation of the technological approaches for cancer diagnosis. It is discussed that the inherent augmented properties of nanostructures enhance the sensitivity of the detection of EVs. It is concluded by reviewing recent integrations of nanostructured‐based optical readouts with diverse machine learning models as novel analysis ventures that can potentially increase the capability of the methods to the point of translation into diagnostic applications.

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A PDMS-based biochip with integrated sub-micrometre position control for TIRF microscopy of the apical cell membrane

Cited by 18 publications

References 42 publications

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates

Morphological plasticity of bacteria—Open questions

Nanostructured‐Based Optical Readouts Interfaced with Machine Learning for Identification of Extracellular Vesicles

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