Axenic stress leads the minor stem cell line of Entamoeba histolytica to defective mitosis and aberrant reversible endopolyploidy Vladimir F. Niculescu* D-86420 Diedorf, Germany, Kirschenweg 1 e-mail:
[email protected]
1. BACKGROUND
2. AUTONOMOUS TERMINAL DIFFERENTIATION
Insufficient knowledge of the cell biology of Entamoeba and its restriction to the axenic way of life led, in the last years, to some inaccurate assumptions. These are: (i) trophozoites of E. histolytica are not capable to produce orderly cysts, (ii) the multinucleated polyploidy of E. histolytica would be the result of S-phase decoupling from cytokinesis, (iii) checkpoints preventing DNA reduplication may be missing in E. histolytica and (iv) multinucleated polyploid cells may be of necessity the precursors of tetranucleated cysts.
I described the stem cell lineage of Entamoeba in detail in my recent work “The stem cell biology of the protist pathogen Entamoeba invadens in the context of eukaryotic stem cell evolution” (see Researchgate). “Spontaneous” ATD cysts are formed in OCB cultures by secondary MAS cells. MAS cells are mitotic arrested cells produced by the minor secondary stem cell line s-SRL; this consists of secondary self renewing SRS cells. MAS cells are the single amoebic cells withdrawing mitotic cell cycle to switch into endocycles.
In contrast, trophozoites of E. histolytica and E. invadens grown under xenic conditions are able to encyst “spontaneously” by autonomous terminal differentiation (ATD); they form mature tetranucleated ATD cysts.These xenic cultures contain multilined cell populations consisting of a dominant cyst-free subpopulation and a minor cell subpopulation producing ATD cysts.
ATD ITD
3. THE STEM CELL LINEAGE OF E. INVADENS (LIFE CYCLE)
4. DEVELOPMENTAL AND NON DEVELOPMENTAL POLYPLOIDIZATION
M A1
t-S V
III
t-
SR
L
MAS ATD
ITD
(Niculescu VF. The stem cell biology of the protist pathogen Entamoeba invadens in the context of eukaryotic stem cell evolution. Stem Cell Biol Res. 2015; 2:2. http://dx.doi.org/ 10.7243/2054-717X-2-2).
P/S
L
ISH cells
MAT
II MAP (RSC)
SR
IV
P/T
I
p -S R L
RL
A2
s-
M, metacyst (hatched innercyst cell), A, amoebulae. Green represents the moderate hypoxic stage in the life cycle (MH), yellow the most oxygenic stage (MO) and red the strong hypoxic stage (SH) of the stem cell lineage as observed in OCB culture sediments. The life cycle of E. invadens consists of three self renewing stem cell lines: the primary line p-SRL, the secondary line s-SRL and the tertiary line t-SRL. Each stem cell line that consists of cycling cells (D1 cells) produces differentiated D2 cells (mitotic arrested cells, MA cells). Primary MAP cells and tertiary MAT cells are reversible differentiated cells capable of cell cycle reentry. Secondary MAS cells are irreversibly committed to endopolyploidization and terminal differentiation producing ATD cysts by autonomous terminal differentiation (“spontaneous” cysts). MAT cells are long living quiescent cells capable of producing ITD cysts, when induced in strong hypoxic/hypoosmotic, non-nutrient media. P/S and P/ T are alternative conversion events that depend on the p02 value of the culture sediment. S/T conversion occured when the MO niche become less oxygenic. RSC, reserve primary stem cells. I-V, check points.
S/T
s-
In contrast to the developmentally endoreplication performed by MAS cells (cells exiting mitotic cycle), the vegetative endopolyploidization observed in axenic cultures of E. histolytica occurs in cycling cells by defective mitosis. Consequently, they are two different states of endopolyploidization: a developmental process linked to terminal differentiation (encystment) and a process of defective mitosis (endomitosis) not related with terminal differentiation. Axenic disorders begin by abnormal centrosome amplification. It gives rise to an abnormal number of centrosomes (instead of normal centrosomes duplication) and dictates the formation of multiple spindle poles [1, 2]. Multipolar spindles lead to multinucleated polyploids with a random ploidy of 6N-14N. These endomitotic polyploids have mechanisms of genome segregation and reductive mitosis (ploidy reversal).
L R S
5. ASYMMETRIC CELL FATE
7. SYMMETRIC CELL FATE
The s-SRL stem cell line of E. invadens in xenic OCB cultures. Asymmetric cell division produces non-identical daughter cells.
The minor cell line of E. histolytica in axenic cultures. It gives rise to identical daughter cells (symmetric cell division)
D1
2
D11
s-SRL
D22
D21
s-SRL, secondary stem cell line; D1 cells, cycling self-renewing daughter cells (SRS cells); D2 cells, mitotic arrested daughter cells withdrawing mitotic cycle (MAS cells)
Logarithmic cell growth, identical progeny (according to Mukherjee et al 2008, 2009)
6. MAS CELLS: MITOTIC EXIT & ATD ENCYSTMENT
8. POLYPLOIDIZATION BY DEFECTIVE MITOSIS
S
G1
o am
p-SRL
ENDOPOLYPLOID CYCLE MAS cells (D2) eb ula e
encystment check point
MITOTIC CYCLE SRS cells (D1)
SRS
G2
M
m ran ulti do n m
ted lea loids uc olyp p
D1
MAS
ATD pr ce
or urs ec ll
D2
cleated u n no lyploid o m C po 8
m on juv onucl eni eated le cy st
M
Axenic cultures lead the minor cell line of E. histolytica to mitotic disorders and random polyploidization
G2
ENDOMITOTIC CYCLE
d e t a e l tetranuc st y c e r matu
S
G1
excystment check point ATD, autonomous terminal differentiation, ATD precursor cell (“precyst”)
Delayed cell division and random nuclear segregation give rise to mononucleated and binucleated polyploids. Synchronization by serum deprivation and serum readdition leads polyploid cells to reductive cell division, ploidy reversal and formation of monoploid (haploid) daughter cells [1, 2].
9. CONCLUSIONS Extrinsic cues (axenic stress) disturb in E. histolytica the mitotic fidelity of the minor cell line. Endomitosis (defective mitosis) leads to non-developmental endopolyploidy respectively multinucleated polyploids unrelated to encystment. Similar disorders do not take place neither in the dominant cell line nor in the minor cell line grown under xenic conditions. All these cells perform normal mitosis.
Reference: 1. Mukherjee C., Clark C.G., Lohia A. 2008 Entamoeba shows reversible variation in ploidy under different growth conditions and between life cycle phases. PLoS Negl Trop Dis. 2:281. (doi: 10.1371/journal.pntd.0000281) ; 2. Mukherjee C, Majumder S, Lohia A. 2009 Inter-Cellular Variation in DNA Content of Entamoeba histolytica Originates from Temporal and Spatial Uncoupling of Cytokinesis from the Nuclear Cycle. PLoS Negl Trop Dis. 3(4): e409 (doi: 10.1371/journal.pntd.0000409)
