Mitochondria organize their genome in protein-DNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and fluorescence microscopy to show the nonspecific DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the flexibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specific transcription regulation by TFAM. This apparent connection between transcription and DNA organization clarifies how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time.

Summary The segregation of the genetic material during mitosis is coordinated by the mitotic spindle, whose mechanism of action depends upon the polarity patterns of its constituent microtubules (MTs)[1, 2]. Homotetrameric mitotic kinesin-5 motors are capable of crosslinking and sliding adjacent spindle MTs [3-11], but it is unknown if they, or other motors, contribute to the establishment of these MT polarity patterns. Here we explored if the Drosophila embryo kinesin-5, KLP61F, which is thought to crosslink both parallel and anti-parallel MTs [7, 12], displays a preference for the parallel or anti-parallel orientation of MTs. In motility assays, KLP61F was observed to crosslink and slide adjacent MTs, as predicted. Remarkably, KLP61F displayed a three-fold higher preference for crosslinking MTs in the antiparallel, relative to the parallel orientation. This polarity preference was observed in the presence of ADP or in ATP plus AMPPNP, but not in AMPPNP alone, which induces instantaneous rigor binding. Also, a purified motorless tetramer containing the C-terminal tail domains displayed an antiparallel orientation preference, confirming that motor activity is not required. The results suggest that, during the morphogenesis of the Drosophila embryo mitotic spindle, the crosslinking and sliding activities of KLP61F could facilitate the gradual accumulation of KLP61F within antiparallel interpolar (ip) MTs at the equator, where the motor could then generate force to drive poleward flux and pole-pole separation.

Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.