Inferring Developmental Stage Composition from Gene Expression in Human Malaria

In the current era of malaria eradication, reducing transmission is critical. Assessment of transmissibility requires tools that can accurately identify the various developmental stages of the malaria parasite, particularly those required for transmission (sexual stages). Here, we present a method for estimating relative amounts of Plasmodium falciparum asexual and sexual stages from gene expression measurements. These are modeled using constrained linear regression to characterize stage-specific expression profiles within mixed-stage populations. The resulting profiles were analyzed functionally by gene set enrichment analysis (GSEA), confirming differentially active pathways such as increased mitochondrial activity and lipid metabolism during sexual development. We validated model predictions both from microarrays and from quantitative RT-PCR (qRT-PCR) measurements, based on the expression of a small set of key transcriptional markers. This sufficient marker set was identified by backward selection from the whole genome as available from expression arrays, targeting one sentinel marker per stage. The model as learned can be applied to any new microarray or qRT-PCR transcriptional measurement. We illustrate its use in vitro in inferring changes in stage distribution following stress and drug treatment and in vivo in identifying immature and mature sexual stage carriers within patient cohorts. We believe this approach will be a valuable resource for staging lab and field samples alike and will have wide applicability in epidemiological studies of malaria transmission

LKB1 Destabilizes Microtubules in Myoblasts and Contributes to Myoblast Differentiation

Background: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings: We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMPactivated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions: Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeleta

Overexpression of LKB1 suppresses microtubule assembly, and LKB1 RNAi increases it.

<p>(A) C2C12 cells were infected with a control adenovirus (control AV) or an adenovirus that drives expression of wild type human LKB1 (LKB1 AV) for 24 hours. Cells were treated with Nocodazole for 1 hour followed by a 7 minute washout, fixation, and microtubule immunofluorescence. Representative microtubule asters are shown. (B) Quantification of aster diameter for A, expressed as mean diameter+/−s.e.m. (C) C2C12 were cells transfected with a control siRNA or an siRNA that targets expression of LKB1 and treated with Nocodazole for 1 hour followed by a 2 minute washout, fixation, and microtubule immunofluorescence. Representative asters are shown. (D) Quantification of aster diameter for C. At least two experiments were done for each condition. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031583#pone-0031583-g004" target="_blank">Fig. 4</a> for LKB1 levels.</p

LKB1 levels and substrate activation increases, and LKB1 redistributes to the cytoplasm during differentiation.

<p>(A) Western blotting was done on samples from the indicated days of differentiation. Myosin heavy chain (myosin) expression increases progressively. LKB1 levels increase by day 1 and peak at day 2 of differentiation. Levels of the phosphorylated forms of LKB1 substrates AMPK and MARK increase by day 1. Tubulin is shown as a loading control. (B, C) Cells were transfected with GFP-LKB1 as described in text. Representative images from undifferentiated cells (B) and cells cultured in differentiation media for three days (C) are shown. The mean ratio of nuclear to cytoplasmic fluorescence was 3.0 in undifferentiated cells and 1.2 in differentiated cells. Bars, 50 µm.</p

Myoblasts show a transient reduction in stable microtubules prior to an ultimate increase in microtubule stabilization.

<p>Cells were differentiated by serum switch for the indicated times, fixed, and immunofluorescence for detyrosinated (glu-) tubulin, a marker of stabilized microtubules, and tyrosinated microtubules, a marker of more dynamic microtubules, was performed. Upper panel shows detyrosinated tubulin, which was reduced at the 24 hour time point and then progressively increased over the next two days. Short linear structures visible with this antibody at 24 hours are primary cilia. Bottom panel shows glu- and tyrosinated tubulin immunofluorescence merged with DNA staining. Bar, 10 µm.</p

LKB1 overexpression accelerates differentiation.

<p>(A) Phase contrast pictures of C2C12 cells uninfected (no virus), infected with an adenovirus overexpressing CRE recombinase and GFP (control AV), or infected with an adenovirus expressing human LKB1, and grown in differentiation media for the indicated number of days. Cells with LKB1 overexpression showed enhanced differentiation. (B) Western blotting shows increased myosin upon LKB1 overexpression. Samples from the indicated days were probed for LKB1, myosin heavy chain (myosin), and phosphorylated AMPK; tubulin, actin, and GAPDH were probed as loading controls.</p

Model.

<p>Myoblasts contain a radial microtubule array that is a mixture of dynamic microtubules (identified in vivo by the presence of a C-terminal tyrosine on alpha-tubulin; depicted here as thin green lines) and stabilized microtubules (identified by post-translational detyrosination; depicted here as thick green lines). Fully differentiated myotubes show a linear microtubule array consisting of abundant detyrosinated/stable microtubules. Our data shows that simple microtubule stabilization blocks the formation of myotubes, and a transient decrease in microtubule stabilization precedes cell elongation and fusion into myotubes. We propose a model in which transient microtubule destabilization facilitates microtubule reorganization, and this is then followed by microtubule stabilization. We suggest that LKB1 plays a role in this microtubule destabilization and/or reorganization, which accounts for its role in the differentiation process.</p

LKB1 RNAi reduces differentiation.

<p>(A) Phase contrast pictures of C2C12 cells transfected an siRNA directed against mouse LKB1 (LKB1 RNAi) and controls were and grown in differentiation media for the indicated number of days. (B) Immunofluorescence for myosin and DNA staining done at day 3 of differentiation show reduced myosin expression in cells with LKB1 RNAi as compared to controls. (C) Western blotting shows reduced myosin expression upon LKB1 RNAi, as well as reduced phosphorylated AMPK (phosho-AMPK) and phosphorylated MARK (phospho-MARK). Tubulin and GAPDH were probed as loading controls.</p