The actin subfamily PtAct4, out of many subfamilies, is differentially localized for specific local functions in Paramecium tetraurelia cells

Abstract

Paramecium tetraurelia possesses more actin isoforms than most other cells. With monospecific antibodies against actin subfamily 4 members, we could label cleavage furrow, nascent food vacuoles, oral apparatus, cilia, cell surface and macronucleus. Expression as green fluorescent protein- (GFP-) fusion protein now allowed us to localize more stringently actin4, e.g., in the macronucleus, particularly when enhanced with anti-GFP antibodies. Posttranscriptional gene silencing of actin4 resulted in disturbances at sites where actin4 has been localized. Cell division was impaired already early on, occasionally resulting in deformed cells. Both micro- and macronuclear development during vegetative cell fission were disturbed. Over longer periods, actin4 silencing entailed reduced phagocytotic activity, paralleled by accumulation of "acidosomes" (late endosomes) near the cytopharynx where they normally fuse with nascent phagosomes. In addition, near the cell surface, extensively misshapen "terminal cisternae" (early endosomes) occurred. In deformed cells, both constitutive endocytosis and stimulated trichocyst exocytosis were impaired. Thus, actin4 exerts pleiotropic effects at widely different sites of the Paramecium cell and disturbances generally coincide with sites where actin4 is normally enriched. Evidently the loss of actin4 cannot easily be compensated for by any other of the large number of actin isoforms occurring in a Paramecium cell.

Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies inParameciumspecies

“…Of a possible 14 paralogues in these three clades, we injected 11 and were able to detect GFP signal from 7 Rab11-GFP fusion proteins in cells. Subcellular localization patterns were determined in live cells using confocal microscopy and verified both by the highly characteristic morphology of structures, such as the oral apparatus and contractile vacuoles in Paramecium cells (Allen, 2000; Sehring et al , 2010), and colabeling with a subcellular marker dye, LysoTracker (Figure S5 and Supplemental Video S2). The identification of structures by characteristic morphology was conducted using simultaneously captured differential interference contrast images (see Supplemental Video S1 for a representative movie).…”

“…This high number of retained duplicate genes from P. aurelia –specific WGDs (and, possibly, the older WGDs) within the species complex represents a host of candidate intermediates on the path to functional change. There is also evidence that at least some WGD1 paralogues in the P. aurelia complex have diverged in function (Schilde et al , 2010; Sehring et al , 2010; Osin´ska et al , 2011). …”

Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics

“…Support comes from the facts that ( i ) mechano‐channels are consistently considered associated with the cytoskeleton, notably F‐actin, in other systems (Lapatsina et al, ), this association being mutually interdependent (Kuipers, Middelbeek & van Leeuwen, ); ( ii ) special cortical isoforms, e.g. Pt Act4, have been identified in P. tetraurelia (Sehring et al, ), and ( iii ) subplasmalemmal localisation of actin was shown by immuno‐electron microscopy (Kissmehl et al, ). Therefore, in principle, F‐actin associated with the cell membrane is available for amplifying gravity signals.…”

Evolutionary Cell Biology of Proteins from Protists to Humans and Plants

“…Pt Act4 does not only occur in the cortex but is also required for initiating formation of phagosomes and of the cleavage furrow (Sehring et al. ). In Paramecium , para‐/ohnologs evidently may serve for vesicle fusion or budding as well as for cyclosis and to regulate access of vesicles to other membranes.…”

“…Importantly, localization differs for the different actin paralogs of Paramecium (Sehring et al. , ). It may also indicate different binding to other proteins and different polymerization kinetics—predictions to be scrutinized.…”