Hetero-regulatory modules inference results.

<p>(A) The predicted hetero-regulatory modules recover known MAPK pathways in <i>S.cerevisiae</i>, including filamentous growth (FG) pathway, mating pheromone (MP) pathway, cell wall integrity (CWI) pathway, and high osmolarity glycerol (HOG) pathway. (B) Distribution of function of HeR module's target genes. The size of the pie, which represents the functional distribution of the corresponding target gene set, is proportional to the number of target genes in the module. (C) Analysis of the target genes of HOG kinase (Ssk2, Pbs2, Hog1) related HeR modules reveals cross-talk between HOG pathway and other MAPK pathways, and indicates potential role of Sok2 in HOG pathway. (D) HeR modules related to transcription factors Tec1 and Ste12 inferred a feedback loop in mating pathway. Shown is the logic of the inference.</p

Co-function prediction using different datasets suggests distinct regulatory pattern in phosphorylation network and transcriptional network.

<p>Shown is the fold change of prediction accuracy using different datasets compared with random levels (the fraction of co-function gene pairs in relevant network). (A) Comparison in phosphorylation networks, KPFN (functional network derived from a microarray study of kinase/phosphatase single deletion strains), KBN (biochemical network derived from in vitro protein chip), and KPIN (physical network of kinase/phosphatase interaction). (B) Comparison in transcriptional regulatory networks, TFBN (transcription factor binding network derived from ChIP-chip experiments) and TFFN (functional networks derived from transcription factor single deletion strains). (C) A linear regulatory model. Regulators R1 and R2 function in a linear regulatory pathway, and T1 and T2 are their targets. R1 and R2 share similar profiles in functional network, but disparate profiles in physical network. (D) A parallel regulatory model. Regulators R1 and R2 function in a parallel regulatory pathway, and T1 and T2 are their targets. R1 and R2 share similar profiles in physical network. However, they have no interaction in functional networks due to genetic buffering. Grey: unobserved data; Green: functional interaction; Blue: physical interaction; Black: no interaction.</p

Motif enrichment analysis reveals different motif usage in the phosphorylation and transcriptional regulatory networks.

<p>Five regulatory motifs were investigated in three networks, phosphorylation network, transcriptional regulatory network, and the combined network. Node A and B represent the regulators (kinase/phosphatase or transcription factor), and node C represents the target gene. In the combined network, node A represents kinase/phophatase and node B represents a transcription factor. P-values and Z-scores are calculated based on a randomly shuffling process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033160#s4" target="_blank">Materials and methods</a>). For the enriched motifs, an example from the corresponding network is provided.</p

Fus3 inhibits filamentous pathway mainly through inactivating Tec1.

<p>Target genes with known function in the two mating pathway kinase related HeR modules, (KP: Ste7, Ste11, Ste20; TF: Ste12, Tec1) and (KP: Fus3;TF: Ste12, Tec1) are shown. The targets of STEs (Ste7, Ste11, Ste20) are enriched with mating pathway genes (green), while the targets of Fus3 are enriched with filamentous pathway genes (red). Deletion of STEs will lead to down-regulation of mating pathway genes, and most of them could be bound by Ste12 and Mcm1 as expected. Deletion of Fus3 mainly up-regulates filamentous pathway genes, which are binding targets of Ste12 and Tec1. Other two filamentous pathway related genes, Dse2 and Dse4, are down-regulated upon Fus3 deletion, and they are inhibitors of filamentous pathway [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033160#pone.0033160-Doolin1" target="_blank">[32]</a>].</p

Immunofluorescent localization of myosin Va in spermatocytes of <i>E. sinensis</i>.

<p>Actin (green) is visualized with FITC conjugated monoclonal anti-actin, myosin Va (red), and DNA with DAPI (blue). Myosin Va mainly localizes in the cytoplasm (<i>arrows</i> in B, D) in spermatocytes; actin mainly localizes in the nucleus, while the cytoplasm presents weak diffuse staining. The chromatin granules dispersed in the nucleus can be seen (C).</p

Myosin Va is expressed in the testis of <i>E. sinensis</i>.

<p>Immunoblots of myosin Va in <i>E. sinensis</i> testis and brain extracts of ICR male mice probed with the anti-myosin Va polyclonal antibody. Brain extracts of mice known to contain myosin Va serve as positive control. A similar band was observed in extracts of <i>E. sinensis</i> testis. β-actin was used as a loading control. The molecular weight marker is shown at right.</p

Localization of myosin Va at the beginning of late stage during <i>E. sinensis</i> spermiogenesis.

<p>(A–D) Immunofluorescent localization of myosin Va. Myosin Va concentrates in the nucleus (<i>arrows</i> in B and D). (E–F) Immunogold labeling of myosin Va. Prominent myosin Va labeling is present in the nucleus (N), and some associates with the nuclear membrane and the membrane complex (MC) (<i>arrows</i> in E, F), as well as mitochondria (M) (<i>arrowhead</i> in E). E. longitudinal section and F. cross section of the initiation of late stage.</p

Localization of myosin Va at the mid stage during <i>E. sinensis</i> spermiogenesis.

<p>(A–D) Immunofluorescent localization of myosin Va in early mid stage. Myosin Va staining can be seen at the proacrosomal vesicle (PV) (<i>arrows</i> in B, D). Scale bar = 10 µm. (E–F) Immunogold labeling of myosin Va is present in the membrane complex (MC) and the proacrosomal vesicle (PV) membrane at late mid stage (<i>arrows</i> in E, F). E shows longitudinal section and F shows cross section of mid stage spermatid.</p

A model of spermiogenesis in Chinese mitten crab <i>E. sinensis</i>.

<p>(A) At early stage proacrosomal granule (PG) and the endoplasmic reticulum vesicle (EV) distribute in the spermatid cytoplasm around the nucleus (N). PM indicates the plasma membrane. (B–C) At mid stage, proacrosomal granule (PG) and the endoplasmic reticulum vesicle (EV) aggregate into proacrosomal vesicle (PV) (B), subsequently, the nucleus (N) initiates to wrap up proacrosomal vesicle (PV) and the membrane complex (MC) emerges between proacrosomal vesicle (PV) and the nucleus (N) (C). The spermatid discards most of the cytoplasm (C). (D–F) At late stage, the proacrosomal vesicle (PV) invaginates to form the acrosomal tubule (AT) (D, E). The mature spermatozoon consists of acrosome with apical cap (AC), acrosomal tubule (AT) and three layers (fibrous layer FL, middle layer ML and lamellar structures LS), centriole (C) at the base of acrosomal tubule (AT), the nuclear cup with several radial arms (RA) and mitochondria (M) (F).</p

Localization of myosin Va at the late stage during <i>E. sinensis</i> spermiogenesis.

<p>(A–D) Immunofluorescent localization of myosin Va. Myosin Va distributes in the nucleus (N) (<i>arrowhead</i> in B,D) and acrosomal tubule (AT) (<i>arrow</i> in B,D), actin staining is present in the nucleus (N) (<i>arrowhead</i> in A,D) and acrosomal tubule (AT) (<i>arrow</i> in A,D), myosin Va colocalizes with actin (D). (E–G) Immunoelectron microscopy analyses of myosin Va. (E) When the acrosomal tubule (AT) begin to form, myosin Va associates with the nuclear membrane and the membrane complex (MC) (<i>arrow</i> in E). (F) After the acrosomal tubule (AT) formation, myosin Va bounds to the nuclear membrane and localizes in the membrane complex (MC) (<i>arrows</i> in F) and acrosomal tubule (AT). (G) Myosin Va localization in mature spermatozoon. Myosin Va distributes in the nucleus (N) and acrosomal tubule (AT). Mitochondria (M) are decorated with myosin Va labeling (<i>arrowheads</i> in E–G).</p