Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca²⁺ signaling in ICC-SM and the relationship between ICC-SM Ca²⁺ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca²⁺transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca²⁺ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca²⁺ entry through voltage-dependent Ca²⁺ conductances, L- and T-type Ca²⁺ channels. Removal of Ca²⁺ from the external solution abolished CTCs. Ca²⁺ release mechanisms reduced the duration and amplitude of Ca²⁺ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca^2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

Generating mammalian cells with specific mtDNA-nDNA combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.