Altered N-glycosylation modulates TgrB1/TgrC1-mediated development but not allorecognition in Dictyostelium

Abstract

Cell surface adhesion receptors play diverse functions in multicellular development. In Dictyostelium, two immunoglobulin-like adhesion proteins, TgrB1 and TgrC1, are essential components with dual roles in morphogenesis and allorecognition during development. TgrB1 and TgrC1 form a heterophilic adhesion complex during cell contact and mediate intercellular communication. The underlying signaling pathways, however, have not been characterized. Here, we report on a mutation that suppresses the tgrB-tgrC1-defective developmental arrest. The mutated gene alg9 encodes a putative mannosyl transferase that participates in N-linked protein glycosylation. We show that alteration in N-linked glycosylation, caused by an alg9 mutation with a plasmid insertion (alg9 ins ) or tunicamycin treatment, can partially suppress the developmental phenotypes caused by tgrC1 deletion or replacement with an incompatible allele. The alg9 ins mutation also preferentially primed cells toward a stalk-cell fate. Despite its effect on development, we found that altered N-linked glycosylation had no discernable effect on TgrB1-TgrC1-mediated allorecognition. Our results show that N-linked protein glycosylation can modulate developmental processes without disturbing cell-cell recognition, suggesting that tgrB1 and tgrC1 have distinct effects in the two processes.

Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium

“…Compared to other mutagenesis methods, such as REMI (Kuspa and Loomis 1992), antisense RNA (Spann et al 1996), and RNA interference (Kuhlmann et al 2006), our method provides a broader spectrum of mutations and opens the field to exploration of modifications that are not necessarily null or loss-of-function mutations. For example, NTG mutagenesis can generate temperature sensitive alleles (Loomis 1969;Liwerant and Pereira Da Silva 1975), and we found strong suppressors of the tgrB1-tgrC1 mismatch phenotype, which were not found by REMI screens (Li et al 2015;Wang and Shaulsky 2015). Some mutations, such as the dominant alleles of tgrB1, cannot be produced by REMI.…”

“…1C; Hirose et al 2011). Previous REMI mutagenesis screens yielded small numbers of weak suppressors (Li et al 2015;Wang and Shaulsky 2015), so we did not know if it was even possible to generate strong suppressors, let alone the relevant target size. We mutated these cells, allowed the population to develop, and selected for spores by detergent treatment.…”

“…Secondly, Dictyostelium has a very gene-rich, haploid genome of 34 Mb (Eichinger et al 2005), which is readily amenable to high-throughput sequencing technologies, especially with the low cost afforded by sample multiplexing. Lastly, we found that REMI suppressors are rare and weak in previous screens for suppressors of the tgrC1-defective phenotypes (Li et al 2015;Wang and Shaulsky 2015), and we wanted a method that would generate a variety of stronger suppressor mutations.…”

The polymorphic proteins TgrB1 and TgrC1 function as a ligand-receptor pair in Dictyostelium allorecognition

“…The transition from unicellular to multicellular development requires interactions between matching TgrB1 and TgrC1. Cells that carry mismatched tgrB1 and tgrC1 alleles behave like the tgrC1null strain (Li et al, 2015). Genetic screens for suppressors of the tgrB1-tgrC1 mismatch or tgrC1 deletion suggested that TgrB1 and TgrC1 participate in signal transduction and that allorecognition and development are mediated by partly overlapping but distinct pathways (Li et al, 2015(Li et al, , 2016Wang and Shaulsky, 2015).…”

Polar pattern formation induced by contact following locomotion in a multicellular system

“…A knockout mutant that fails to express the cell–cell adhesion molecule TgrB1 exhibits reduced CFL activity(Fujimori et al, 2019). TgrB1 is known to mediate cell–cell adhesion via a heterophilic interaction with its partner TgrC1(Fujimori et al, 2019; Hirose et al, 2011; 2015; C.-L. F. Li et al, 2015). We first assessed whether the tgrb1 null mutant forms propagating bands.…”