Direct evidence of amyloid precursor–like protein 1 <i>trans</i> interactions in cell–cell adhesion platforms investigated via fluorescence fluctuation spectroscopy

Dunsing, Valentin; Mayer, Magnus C.; Liebsch, Filip; Multhaup, Gerhard; Chiantia, Salvatore

doi:10.1091/mbc.e17-07-0459

Cited by 35 publications

(50 citation statements)

References 59 publications

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“…In conclusion, the data currently available suggest that APLP2 dimerization is only moderately influenced by metal ion binding, whereas APP and APLP1 are affected more strongly. Moreover, APP trans-dimerization is modulated mainly by copper, whereas zinc affects predominantly APLP1 trans-dimerization (Baumkotter et al 2014;Mayer et al 2016;Dunsing et al 2017). Here, we demonstrate that copper promotes also APLP1 and APLP2 trans-dimerization and that zinc attenuates the trans-directed dimerization of APP, likely via a novel high-affinity zinc binding site in the E1 domain.…”

mentioning

confidence: 64%

“…6). Zincinduced trans-interaction of APLP1 has been reported before (Dunsing et al 2017), depending on a newly identified zinc binding site in the E2 domain of APLP1 (Mayer et al 2014(Mayer et al , 2016. It has been assumed that increased trans-interaction of APLP1 might be because of zinc-induced oligomerization of APLP1 within the plasma membrane, involving alterations in the submembraneous cytoskeleton.…”

Section: Discussionmentioning

confidence: 96%

“…) and favor trans‐directed dimerization (Dunsing et al . ). However, similarly as performed in cell culture studies before for copper binding in the E1 domain (Baumkotter et al .…”

Section: Discussionmentioning

confidence: 97%

“…Zinc‐induced trans‐interaction of APLP1 has been reported before (Dunsing et al . ), depending on a newly identified zinc binding site in the E2 domain of APLP1 (Mayer et al . , ).…”

Section: Discussionmentioning

confidence: 99%

“…; Dunsing et al . ). Here, we demonstrate that copper promotes also APLP1 and APLP2 trans‐dimerization and that zinc attenuates the trans‐directed dimerization of APP, likely via a novel high‐affinity zinc binding site in the E1 domain.…”

mentioning

confidence: 97%

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Copper and zinc ions govern the trans‐directed dimerization of APP family members in multiple ways

August

Schmidt

Klingler

et al. 2019

Journal of Neurochemistry

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The amyloid precursor protein (APP) and its homologs amyloid precursor‐like protein 1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans‐directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growth factor‐like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans‐dimerization. Using isothermal titration calorimetry, co‐immunoprecipitation, and in vitro bead aggregation assays, we show that copper promotes cis‐ as well as trans‐directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP‐specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis‐ but trans‐dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper‐induced trans‐directed dimerization of APP. Together, we identified a high‐affinity APP‐specific zinc binding site in the E1 domain and revealed contrasting cis‐ and trans‐directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo‐ and heterotypic trans‐cellular APP/APLPs complexes.

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mentioning

confidence: 64%

Section: Discussionmentioning

confidence: 96%

“…) and favor trans‐directed dimerization (Dunsing et al . ). However, similarly as performed in cell culture studies before for copper binding in the E1 domain (Baumkotter et al .…”

Section: Discussionmentioning

confidence: 97%

“…Zinc‐induced trans‐interaction of APLP1 has been reported before (Dunsing et al . ), depending on a newly identified zinc binding site in the E2 domain of APLP1 (Mayer et al . , ).…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 97%

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Copper and zinc ions govern the trans‐directed dimerization of APP family members in multiple ways

August

Schmidt

Klingler

et al. 2019

Journal of Neurochemistry

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Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS)

Mørch¹,

Schneider²

2023

Methods in Molecular Biology

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Activation of immune cells and formation of immunological synapses (IS) rely critically on the reorganization of the plasma membrane. These highly orchestrated processes are driven by diffusion and oligomerization dynamics, as well as by single molecule interactions. While slow macro- and meso-scale changes in organization can be observed with conventional imaging, fast nano-scale dynamics are often missed with traditional approaches, but resolving them is, nonetheless, essential to understand the underlying biological mechanisms at play. Here, we describe the use of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) to study reorganization and changes in molecular diffusion dynamics and interactions during IS formation and in other biological settings. We focus on the practical aspects of the measurements including calibration and alignment of the optical setup, present a comprehensive protocol to perform the measurements, and provide data analysis pipelines and strategies. Finally, we show an exemplary application of the technology to studying Lck diffusion during T-cell signaling.

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Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems

et al. 2018

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Fluorescence correlation spectroscopy (FCS) is a powerful tool to quantitatively study the diffusion of fluorescently labeled molecules. It allows in principle important questions of macromolecular transport and supramolecular aggregation in living cells to be addressed. However, the crowded environment inside the cells slows diffusion and limits the reservoir of labeled molecules, causing artifacts that arise especially from photobleaching and limit the utility of FCS in these applications. We present a method to compute the time correlation function from weighted photon arrival times, which compensates computationally during the data analysis for the effect of photobleaching. We demonstrate the performance of this method using numerical simulations and experimental data from model solutions. Using this technique, we obtain correlation functions in which the effect of photobleaching has been removed and in turn recover quantitatively accurate mean-square displacements of the fluorophores, especially when deviations from an ideal Gaussian excitation volume are accounted for by using a reference calibration correlation function. This allows quantitative FCS studies of transport processes in challenging environments with substantial photobleaching like in living cells in the future.

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Direct evidence of amyloid precursor–like protein 1 trans interactions in cell–cell adhesion platforms investigated via fluorescence fluctuation spectroscopy

Cited by 35 publications

References 59 publications

Copper and zinc ions govern the trans‐directed dimerization of APP family members in multiple ways

Copper and zinc ions govern the trans‐directed dimerization of APP family members in multiple ways

Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS)

Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems

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