Targeted GECO Calcium Assays for Sub-cellular Domains
Mitochondria Calcium Localization
MTCaL
- 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.
The sensor reports mitochondrial calcium in response to the addition of carbachol. HEK293 cells were transducer with the mitochondrial calcium sensor and stimulated with carbachol or vehicle control and read fluorescence using a Synergy MX standard plate reader.
MTCaL 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 MTCaL 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.
Videos
Time lapse imaging of mitochondrial Ca2+ in AD293 Cells. MTCaL 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 MTCaL 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 niclosamide, 10µM, right.
Vehicle (DMSO), left, and FCCP, 10µM, right.
Mitochondrial Ca2+
Simultaneous measurement of mitochondria Ca2+ and cell motility in response to small molecule treatment. AD293 were transduced with MTCaL and imaged on the EVOS M7000 every 4 minutes for several hours. Compounds were added at t=0: DMSO (Vehicle) spiperone (10µM), niclosamide (10µM) and FCCP (10µM).
Top Row: Mitochondrial Ca2+ reported by MTCaL mNeonGreen Ca2+ sensor from segmented cells using FIJI.
Cell Motility
Bottom Row: Cell motility was quantified in FIJI as the frame to frame displacement of the mean position of the H2B red nuclear marker across the entire imaging field, expressed relative to vehicle control. On this scale, 0 indicates vehicle matched movement, and -1 indicates no detectable cell movement. Spiperone evoked a rapid transient rise in mitochondrial Ca2+ that peaked within 4 minutes and decayed to a sustained plateau, coupled with a modest reduction in movement. Niclosamide produced a small, transient increase in the mitochondrial Ca2+ signal that returned to baseline, together with a rapid loss of movement that reached the detection floor within 30 minutes and persisted for the remainder of the recording. FCCP produced a larger rise in mitochondrial Ca2+ signal followed by a sustained plateau above baseline, and a comparably rapid and sustained loss of movement.
TRPML1-Lysosomal-Ca2+ sensor (TLC) for the Ca2+ Dyshomeostasis Neurodegeneration Model
Here we present a genetically encoded TRPML1 Lysosomal Ca2+ (TLC) sensor that enables direct measurement of Ca2+ release with high specificity. Using this tool we show that small molecule agonists differ in their ability to selectively activate TRPML1, with MSK-83 demonstrating higher specificity compared to ML-SA5. The Assay provides a powerful approach for interrogating TRPML1 function in human cells and for identifying modulators of lysosomal Ca2+ signaling.
Perturbed Ca2+ signaling leads to synaptic loss, amyloid and tau aggregation, mitochondrial dysfunction, lysosomal alkalinization, and superoxide production, all of which are hallmarks of neurodegenerative diseases. Evidence from Alzheimers models suggest that ER-derived Ca2+ signals disrupt mitochondria-lysosome interactions, further exacerbating cellular stress.
