MitoMatrixCaL
Mitochondrial Calcium Sensor

Mitochondria Calcium Localization; MitoMatrixCaL

Mitochondrial Ca²⁺ handling sits at the center of some of the most fundamental operations in cell biology including energy metabolism, apoptosis, reactive oxygen species (ROS) production, and cell signaling. Each pathway is affected by, and influences how mitochondria take up, buffer, and release calcium. Mitochondrial Ca²⁺ handling is central to many disease pathways and  the Mitochondria Calcium sensor (MitoMatrixCaL) is built to enable researchers to examine Ca²⁺ dynamics in real time. MitoMatrixCaL lets you visualize mitochondrial Ca²⁺ uptake kinetics in response to physiological or pharmacological stimuli over time. 

• Genetically encoded — Can be expressed in many in cell types, including primary neurons, iPSC-derived cell types, and immortalized cell lines; no exogenous dye loading or washout steps
• BacMam delivery — fast, efficient, dose-controlled transduction across a broad range of mammalian cell types, including cells that are difficult to transfect
• Live-cell, real-time — track dynamics from seconds or hours to days, rather than a single timepoint in fixed cells
• Mitochondrial matrix–targeted — a COX8 targeting sequence delivers the calcium sensor specifically to the mitochondrial matrix, monitoring a Ca²⁺ pool distinct from cytosolic or ER Ca²⁺ pools
• Built-in nuclear marker — no need to co-transduce a separate reagent for cell identification, segmentation or counting
• Screen in 384 well format or resolve single mitochondria— Same virus, same cells, no reoptimization. Plate reader and microscope compatible.

MitoMatrixCaL sensor was delayed in comparison to the cytosolic Ca²⁺ sensor (in red), which was expected. The initial increase in Ca²⁺ shown by the cytosolic Ca²⁺ sensor is due to the addition of carbachol activating muscarinic subtypes which evoke Ca²⁺ release. Mitochondria then take-up Ca²⁺ freely, as reported by the MitoMatrixCaL sensor. HEK293 cells were transduced with MTCaL or R-GECO (cytosolic Ca²⁺ sensor) and HM1 and stimulated with carbachol or vehicle control (not shown). Data was collected using a Synergy MX standard plate reader.

One reagent that scales from screening using a standard plate reader to looking at individual mitochondrion

Works on both microscope and plate readers

This Spiperone response was easily read using a standard Synergy MX plate reader

Videos

Time lapse imaging of mitochondrial Ca²⁺ in AD293 Cells. MitoMatrixCaL delivers a red nuclear marker and a mitochondria matrix-targeted mNeonGreen Ca²⁺ sensor from a single BacMam construct. Both cell identification and mitochondrial Ca²⁺ can be tracked in the same field. AD293 cells were transduced with MitoMatrixCaL and imaged on an EVOS M7000 at 4 minute intervals over five hours. Small molecules that induce an increase in mitochondrial Ca²⁺ were added at t = 40 minutes. The timescale is shown in the upper left corner of each video

Vehicle (DMSO) left, and spiperone, 10µM right.

Vehicle (DMSO) left, and spiperone, 10µM right. The addition of spiperone raises cytosolic Ca²⁺ through a phospholipase C-dependent ER Ca²⁺ release (Antona et al. 2022) leading to mitochondrial uptake.

Vehicle (DMSO), left, and niclosamide, 10µM, right

Niclosamide impairs the growth of P53-deficient cells (tumor cells) but was originally developed for the treatment of intestinal tapeworm infections. Niclosamide is a hydrogen ionophore that translocates protons across the mitochondrial membrane inducing mitochondrial uncoupling (Kumar et al. 2018).

Vehicle (DMSO), left, and FCCP, 10µM, right

FCCP is a mitochondrial uncoupler that disrupts ATP production by transporting protons across mitochondrial membranes (Benz and McLaughlin 1983).

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