Fig 1.
Genomic evidence for focal adhesion complex components in metazoan and non-metazoan Unikonts.
Results shown are from [8–10, 30], and are based on the genomic analysis of several Metazoa representatives, the Choanoflagellates representatives Monosiga brevicollis and Proterospongia sp., the Filasterea representative Capsaspora owczarzaki, the Ichthyosporea representative Sphaeroforma, several representatives of the Dikarya fungi, the basal fungi representatives Allomyces macrogynus (Blastocladiomycota), Spizellomyces punctatus, and Batrachochytrium dendrobatidis (Chytridiomycota), Orpinomyces sp. C1A, Anaeromyces robustus, Neocallimastix californiae, and Piromyces finnis (Neocallimastigomycota), the Apusozoa representative Amastigomonas sp., and several representatives of the Ameobozoa. The dendogram is not drawn to scale and only serves to show the relationships between the different groups. Cells shaded in black denote clear homologues were identified in all representative genomes, cells shaded in grey denote clear homologues were identified in some but not all representative genomes, and cells shaded in white denote no homologues were identified in any of the representative genomes.
Fig 2.
A simplified schematic of the focal adhesion machinery in Metazoa.
Focal adhesion proteins are color coded as follows: Integrins (ECM receptors) are brown, Scaffolding proteins are green, proteins of the IPP complex are blue, signaling kinases are yellow. F-actin polymers are shown in red and proteins of the extracellular matrix are shown in pink.
Fig 3.
Schematic of the protocol used to collect the different developmental stages of C1A employed for the transcriptional study.
Table 1.
Quantitative PCR primers used for cDNA amplification.
Fig 4.
Maximum likelihood phylogenetic analysis of C1A predicted scaffolding proteins.
All evolutionary analyses and model selections were conducted in MEGA7 [13]. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap values, in percent, are based on 100 replicates and are shown for branches with >50% bootstrap support. Trees are shown for: (A) α-actinin based on the JTT model with a discrete Gamma distribution (variable site γ shape parameter = 2.0327). Analysis involved 21 amino acid sequences, with a total of 535 positions in the final dataset. (B) Paxillin based on the Dayhoff model with a discrete Gamma distribution (variable site γ shape parameter = 1.7755). Analysis involved 12 amino acid sequences, with a total of 535 positions in the final dataset. (C) Talin based on the Le_Gascuel_2008 model with a discrete Gamma distribution (variable site γ shape parameter = 3.0802). Analysis involved 13 amino acid sequences, with a total of 441 positions in the final dataset. (D) Vinculin based on the Le_Gascuel_2008 model with a discrete Gamma distribution (variable site γ shape parameter = 3.4035). Analysis involved 14 amino acid sequences, with a total of 145 positions in the final dataset.
Fig 5.
C1A predicted scaffolding proteins functional domain structure and organization, and predicted protein structure modeling.
Results are shown for (A) α-actinin, (B) talin, and (C) vinculin. For each predicted protein, the first row (I) corresponds to the predicted Pfam domain organization. This is followed by 1–5 rows (II-VI) each corresponding to a functional domain. On the left of each of these rows, secondary structure alignments of C1A predicted domian compared to a template’s known and predicted secondary structure are shown. On the right of each row, predicted tertiary structures of C1A domains are shown in pink, compared to the template’s known tertiary structure in cyan. Superimposed structures are also shown. Row (A-II): predicted C1A α-actinin CH domain structure compared to PDB: 1SJJ from Gallus gallus. Row (A-III): predicted C1A α-actinin spectrin domain structure compared to PDB: 1SJJ from Gallus gallus. Row (A-IV): predicted C1A α-actinin Ca2+ insensitive EF hand domain structure compared to PDB: 1SJJ from Gallus gallus. Row (B-II): predicted C1A talin middle domain structure compared to PDB: 1SJ7 from Mus musculus. Row (B-III): predicted C1A talin VBS1 domain structure compared to PDB: 2L10 from Mus musculus. Row (B-IV): predicted C1A talin VBS2 domain structure compared to PDB: 2KVP from Mus musculus. Row (B-V): predicted C1A talin I/LWEQ domain (Blocks 1–3) structure compared to PDB: 2JSW from Mus musculus. Row (B-VI): predicted C1A talin I/LWEQ domain (Block 4 comprizing the dimerization domain) structure compared to PDB: 2QDQ from Mus musculus. Row (C-II): predicted C1A vinculin domain structure compared to PDB: 1TR2 from Homo sapiens.
Table 2.
Results of C1A scaffolding proteins comparison to the Pfam database, as well as secondary and tertiary structure predictions.
Fig 6.
Transcriptional levels of genes encoding scaffolding proteins in the presence and absence of an extracellular matrix polysaccharide.
The number of transcript copies of talin, paxillin, vinculin, and α-actinin relative to the number of transcript copies of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown when C1A was grown on soluble cellobiose media (☐) (i.e. in absence of an ECM) versus when grown on a MCC media (■) (i.e. in presence of an ECM). Error bars are standard deviations from two experiments (each with 2 replicates) for paxillin, vinculin, and α-actinin, and four experiments (each with two replicates) for talin. Values were significantly higher in absence of ECM for talin (5.7-fold increase, Student t-test P-value = 0.001), α-actinin (13.1-fold increase, Student t-test P-value = 0.009), vinculin (8.7-fold increase, Student t-test P-value = 0.008), and paxillin (5.7-fold increase, Student t-test P-value = 0.07).
Fig 7.
Transcriptional levels of genes encoding scaffolding proteins in various life cycle stages of C1A.
The number of transcript copies of talin, paxillin, vinculin, and α-actinin relative to the number of transcript copies of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown for the active sporangia (☐) versus the late sporangia (■) samples (A), as well as for the flagellated spores (☐) versus germinating spores (■) samples (B). Error bars are standard deviations from two experiments (each with 2 replicates) for paxillin, vinculin, and α-actinin, and four experiments (each with two replicates) for talin. Transcriptional levels were significantly higher in active sporangia compared to late sporangia [for talin (773-fold increase, Student t-test P-value = 0.005), vinculin (807-fold increase, Student t-test P-value = 0.03), paxillin (17,463-fold increase, Student t-test P-value = 0.049), and α-actinin (33-fold increase, Student t-test P-value = 0.007)]. Likewise, transcriptional levels were significantly higher in germinating spores compared to flagellated spores [for talin (730-fold increase, Student T t-test P-value = 0.002), vinculin (19,589-fold increase, Student t-test P-value = 0.022), paxillin (6,300,742-fold increase, Student T-test p-value = 0.018), and alpha-actinin (43,513,108-fold increase, Student t-test P-value = 0.048)]. Comparing active sporangia to germinating spores, transcriptional levels were also significantly higher in germinating spores [for talin (7.8-fold increase, Student t-test P-value = 0.005), vinculin (160-fold increase, Student t-test P-value = 0.027), paxillin (647-fold increase, Student t-test P-value = 0.048), and alpha-actinin (1,494-fold increase, Student t-test P-value = 0.007)].