Figure 3. Predicted sets of kinetoplasts in mutant trypanosomes follwing failure of specified steps of the kinetoplast cycle.

Kinetoplast DNA (kDNA) (hollow red circle) was considered as a substrate for synthesis to produce a double-sized kinetoplast (K_U). Cleavage of a kinetoplast precedes mitosis, and normally produces two equal-sized kDNAs and one nucleus (2K1N) in one trypanosome. Mitosis of 2K1N trypanosomes results in 2K2N cells that can go through cytokinesis and generate 1K1N cells. Early abnormal kinetoplasts predicted for each class of mutant in kinetoplast cycle is illustrated. In all cases sustained proliferation of early mutant trypanosomes leads to an increased fraction of dyskinetoplastic (0K1N) cells, but the path to this end result, as revealed by the panel of abnormal kinetoplasts that accompanies each mutation varies. A, Class I, kDNA Synthesis Mutants. After loss of a protein that is essential for synthesis of kDNA the total amount of DNA per kinetoplast decreases. Hence after scission of that kDNA the progeny has less than one kinetoplast’s equivalent (i.e., Ks) whereas the nucleus has a regular amount of DNA, making the cell a 2K_S1N trypanosome. After mitosis, a 2K_S2N trypanosome emerges which can go through cytokinesis. Following multiple rounds of division, the trypanosome gradually loses all of its kDNA and becomes dyskinetoplastic (i.e., 0K1N). B, Class II, Site of Scission Selection. After synthesis of kDNA in a normal cell a double-size uncleaved kinetoplast (K_U) is the product in a 1K_U1N cell: the kDNA is cleaved normally into two equal-sized progenies, before mitosis. Faulty choice of the site of scission resulting from knockdown of a factor that is import for identification of the scission site will cause cleavage of kDNA into two networks of unequal size (i.e., asymmetric scission of kinetoplast). The larger kinetoplast (K_L) and the smaller kDNA (K_S) can be found in premitotic (1K_S/K_L1N) cells or in post-mitotic 1K_S/K_L2N trypanosomes. Repeated asymmetric kinetoplast scission and proliferation of progeny eventually leads to a population of trypanosomes with a significant fraction of dyskinetoplastics (0K1N). C, Class III, Kinetoplast Scission Factors (KSFs). Cleavage of a double-size kinetoplast (1K_U1N) into two networks is essential for inheritance of kinetoplasts. After knockdown of genes encoding KSFs, 1K_U1N remains uncleaved in premitotic trypanosomes. However, failure of kDNA scission does not foil mitosis, so nuclear division results in 1K_U2N trypanosomes that may accumulate in the population. Cytokinesis of 1K_U2N cells produces 0K1N (dyskinetoplastic) and 1K_U1N cells that after proliferation increase the fraction of dyskinetoplastic trypanosomes. D, Class IV, Separation of cleaved kinetoplasts. After scission of kinetoplasts the kDNA networks are detected by light microscopy as two entities when they move apart. Thus, in mutants where the separation of kinetoplasts does not take place, only one kDNA will be detectable although scission of kinetoplasts has occurred. Kinetoplasts in Class IV mutant 1K2N and 1K1N cells are only distinguishable from those of Class III (KSF) mutants in electron microscopy studies; they will reveal cleaved but adjacent kinetoplasts for separation factors, but show uncleaved kDNA in mutants of KSF’s. E, Class V, Partitioning of Kinetoplasts. During cytokinesis two kinetoplasts from mitotic 2K2N trypanosomes are sorted into two new progeny each of which has a 1K1N organelle content. If partitioning of the two kinetoplasts fails, the progeny trypanosomes are 2K1N and 0K1N. Thus, detection of 0K1N cells in absence of 1K_U2N early after gene knockdown is diagnostic of partition mutants.