Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks

Abstract

Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. In this report, we show that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, we found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, we show that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. We conclude that the human Miltons are important mediators of the mitochondrial trafficking machinery.

Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro

“…Conformational changes in Miro might require the presence of an additional macromolecular binding partner, as seen in some other EF hand proteins [15]. Miro interacts with kinesin-1 [6][7][8], mitofusin [27], Milton [28][29][30], and Displayed is the MiroS-MgGDP structure, with select residues from the aligned apo-MiroS structure shown in orange. GDP is shown as sticks (2Fo-Fc map, 1.2s), and Mg 2 þ as a purple sphere.…”

Trafficking Kinesin Protein (TRAK)-mediated Transport of Mitochondria in Axons of Hippocampal Neurons

“…TRAK2 DN Does Not Homodimerize with Full-length TRAK2-TRAK2 is known to form at least homodimers (14,24). If TRAK2 DN was able to dimerize with full-length TRAK2, this may negate its use as a dominant negative because full-length TRAK2 may retain the ability to bind Miro1/2.…”

“…The association between the TRAKs and KHC is direct and involves interaction between TRAKs and the KHC non-motor, C-terminal cargo binding domain (13). In heterologous expression, TRAKs and Milton are targeted to mitochondria (11,12,14). The co-expression of TRAK1, TRAK2, or Milton with KHC results in the redistribution of mitochondria such that they colocalize with TRAK1, TRAK2, Milton, and kinesin heavy chains at the tips of cellular processes (13,15,16).…”

Assisted reproductive technologies to prevent human mitochondrial disease transmission

“…Mammalian mitochondria associate with each other and with other organelles 63,64 , increasing the likelihood of carryover in MRT. Several reports suggest that mitochondria are interconnected via a network of microtubules 63,65 . It is therefore possible that microtubule-mediated connections could be undone with inhibitors such as nocodazole before MRT, although residual levels of the inhibitor could potentially disrupt chromosome segregation during the first mitotic cleavage, resulting in aneuploidy; the inhibitory effects of nocodazole are reversible, but any damage to, for instance, chromosome-spindle attachments may not be.…”