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Timescale of hole closure during plasma membrane repair estimated by calcium imaging and numerical modeling.

ABSTRACT: Plasma membrane repair is essential for eukaryotic cell life and is triggered by the influx of calcium through membrane wounds. Repair consists of sequential steps, with closure of the membrane hole being the key event that allows the cell to recover, thus identifying the kinetics of hole closure as important for clarifying repair mechanisms and as a quantitative handle on repair efficiency. We implement calcium imaging in MCF7 breast carcinoma cells subject to laser damage, coupled with a model describing the spatio-temporal calcium distribution. The model identifies the time point of hole closure as the time of maximum calcium signal. Analysis of cell data estimates the closure time as: [Formula: see text] s and [Formula: see text] s using GCaMP6s-CAAX and GCaMP6s probes respectively. The timescale was confirmed by independent time-lapse imaging of a hole during sealing. Moreover, the analysis estimates the characteristic time scale of calcium removal, the penetration depth of the calcium wave and the diffusion coefficient. Probing of hole closure times emerges as a strong universal tool for quantification of plasma membrane repair.

SUBMITTER: Klenow MB

PROVIDER: S-EPMC7895973 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Timescale of hole closure during plasma membrane repair estimated by calcium imaging and numerical modeling.

Klenow Martin Berg MB Heitmann Anne Sofie Busk ASB Nylandsted Jesper J Simonsen Adam Cohen AC

Scientific reports 20210219 1

Plasma membrane repair is essential for eukaryotic cell life and is triggered by the influx of calcium through membrane wounds. Repair consists of sequential steps, with closure of the membrane hole being the key event that allows the cell to recover, thus identifying the kinetics of hole closure as important for clarifying repair mechanisms and as a quantitative handle on repair efficiency. We implement calcium imaging in MCF7 breast carcinoma cells subject to laser damage, coupled with a model ...[more]

PMID: 33608587

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Project description:Analysis of transcriptome in moss Physcomitrella patens CNGCb null mutant at 25 and 34 degrees C for 30 minutes. Results provide insight into role of CNGCb in acquired thermotolerance induced by non-lethal heat treatment. Typically at dawn of a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to timely develop a heat-shock response (HSR) and accumulate protective heat shock proteins (Hsps), in anticipation of upcoming harmful temperatures at mid-day. Here, we found that the CNGCb gene from Physcomitrella patens and its Arabidopsis ortholog CNGC2, encode for a component of cyclic nucleotide gated Ca2+ channels acting as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to a HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused altered Ca2+ signaling and a sustained Ca2+ influx. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermo-responsive Ca2+-channels in wild type cells. Deletion of CNGCb led to a total absence of one, and it increased the open probability of the remaining two thermo-responsive Ca2+ channels. Thus, both in Arabidopsis and moss, CNGC2 and CNGCb are expected to form with other related CNGCs, heteromeric Ca2+ channels in the plasma membrane that respond to mild increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance. The WT moss tissues were heat-shocked for a half an hour at 34°C and 38°C and CNGCb at 25and 34°C followed by liquid nitrogen freezing. Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and two biological replicate samples for each treatment, were extracted. An Agilent-certified microarray service lab (MOgene, LC, St. Louis, MO, USA) was used to verify the integrity of the RNA and perform the microarray experiments. Two biological replicates were performed.

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Timescale of hole closure during plasma membrane repair estimated by calcium imaging and numerical modeling.

Publications

Timescale of hole closure during plasma membrane repair estimated by calcium imaging and numerical modeling.

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