A Gene Encoding the Major Beta Tubulin of the Mitotic Spindle in Physarum polycephalum Plasmodia

Abstract

The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a beta-tubulin cDNA clone, beta 105, which is shown to correspond to the transcript of the betC beta-tubulin locus and to encode beta 2 tubulin, the beta tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that beta 2 tubulin is only 83% identical to the two beta tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum beta 2 tubulin and the beta tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum beta 2 tubulin is no more similar to, for example, Aspergillus beta tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged beta tubulin as well as one or more beta tubulins that conform more closely to a consensus beta-tubulin sequence. We suggest that beta-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among beta tubulins could have resulted through neutral drift. For example, exclusive use of Physarum beta 2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the beta tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of beta tubulins may allow positive selection to operate more freely to refine beta-tubulin function.

Spatial transcriptomic and single-nucleus analysis reveals heterogeneity in a gigantic single-celled syncytium

“…For example, certain tubulins and one kinesin – major components of the spindle assembly pathway – are differentially expressed between the plasmodium and amoebae ( Figure 4G ). We find that alpha tubulin 1a (TBAD) and beta tubulin 1 (TBB1) are spindle markers expressed in both states, whereas alpha tubulin 1b (TBAN) and beta tubulin 2 and 4 (TBB2, TBB4) are only detected at high levels in the plasmodium, consistent with previous reports ( Burland et al, 1988 ; Burland et al, 1983 ). Also, the epsilon and gamma tubulin chains (TBE, TBG) involved in centrosome duplication and spindle formation and the spindle-associated kinesin KIF1 are detected at much higher levels in the plasmodium.…”

Microtubules in Microorganisms: How Tubulin Isotypes Contribute to Diverse Cytoskeletal Functions

“…Cells survive disruption of TUB3 but not TUB1 (Schatz et al, 1986b). Northern blots with either gene as probe suggested that TUB1 transcript was more prevalent than TUB3, although probe crosshybridization prevented accurate quantification (Schatz et al, Burland et al (1983), Burland et al (1984), Burland et al (1988); Schedl et al (1984b), Krämmer et al (1985); Singhofer-Wowra et al (1986), Monteiro and Cox (1987a), Monteiro and Cox (1987b), Green et al (1987) 1986a). The idea that cells harbor more Tub1 than Tub3 subunits is consistent with the finding that although lethal, tub1Δ can be rescued by increased expression of TUB3 (Schatz et al, 1986b).…”

“…Chlamydomonas reinhardtii tua1, tua2 tub1, tub2 each isoform: differential anti-tubulin drug resistance Kato- Minoura et al (2020). Physarum polycephalum altA, altB(N), altB(E), betA, betB, betC altA, altB(N), altB(E): MT assembly in plasmodia and myxamoeba Burland et al (1983), Roobol et al (1984), Cunningham et al (1993);Gerber et al (2022), altA builds flagellar MTs via PTM in amoeba and flagellates Green and Dove (1984), Blindt et al (1986), Diggins and Dove (1987), Sasse et al (1987); betA: MT structure assembly during myxamoeba stage Burland et al (1988), Diggins-Gilicinski et al (1989), Gerber et al (2022; betB: constitutively expressed in plasmodia and myxamoeba Werenskiold et al (1988), Paul et al (1989), benzimidazole resistance to both plasmodia and myxamoeba Burland et al (1984); betC: anastral mitotic spindle assembly in plasmodia Burland et al (1983), Roobol et al (1984), Gerber et al (2022), MT assembly in developing cells during amoeboid-plasmodial transition Solnica-Krezel et al (1988). *In Table 2, gene names are presented using organism-specific syntax.…”

“…P. polycephalum , a myxomycete or acellular slime mold, is a protist that harbors a relatively large complement of tubulin isotypes: five α-tubulins [ ALTA , ALTB(N) , ALTB(E) , ALTC , ALTD ] ( Burland et al, 1983 ; Schedl et al, 1984b ; Krämmer et al, 1985 ; Singhofer-Wowra et al, 1986 ; Green et al, 1987 ; Monteiro and Cox, 1987a , Monteiro and Cox, 1987b ) and three β-tubulins ( BETA , BETB , BETC ) ( Table 1 ) ( Burland et al, 1984 ; Singhofer-Wowra et al, 1986 ; Burland et al, 1988 ; Solnica-Krezel et al, 1988 ; Werenskiold et al, 1988 ). This pool of tubulin isotypes is used to support a range of MT-structures and functions in this simple protist.…”

“…Of the three β-tubulin isotypes, BETA and BETC are exclusively expressed and incorporated into MT structures of the myxamoeba and plasmodia stages, respectively ( Burland et al, 1988 ; Diggins-Gilicinski et al, 1989 ; Gerber et al, 2022 ). On the other hand, BETB is constitutively expressed during both the myxamoeba and plasmodia phases ( Werenskiold et al, 1988 ) and incorporated into all MT structures ( Paul et al, 1989 ).…”