Inferring causal molecular networks: empirical assessment through a community-based effort

Inferring molecular networks is a central challenge in computational biology. However, it has remained unclear whether causal, rather than merely correlational, relationships can be effectively inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge that focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results constitute the most comprehensive assessment of causal network inference in a mammalian setting carried out to date and suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess the causal validity of inferred molecular networks

Inferring causal molecular networks: empirical assessment through a community-based effort

It remains unclear whether causal, rather than merely correlational, relationships in molecular networks can be inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge, which focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective, and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess inferred molecular networks in a causal sense

ZYG-9ch-TOG promotes the stability of acentrosomal poles via regulation of spindle microtubules in C. elegans oocyte meiosis.

During mitosis, centrosomes serve as microtubule organizing centers that guide the formation of a bipolar spindle. However, oocytes of many species lack centrosomes; how meiotic spindles establish and maintain these acentrosomal poles remains poorly understood. Here, we show that the microtubule polymerase ZYG-9ch-TOG is required to maintain acentrosomal pole integrity in C. elegans oocyte meiosis. We exploited the auxin inducible degradation system to remove ZYG-9 from pre-formed spindles within minutes; this caused the poles to split apart and an unstable multipolar structure to form. Depletion of TAC-1, a protein known to interact with ZYG-9 in mitosis, caused loss of proper ZYG-9 localization and similar spindle phenotypes, further demonstrating that ZYG-9 is required for pole integrity. However, depletion of ZYG-9 or TAC-1 surprisingly did not affect the assembly or stability of monopolar spindles, suggesting that these proteins are not required for acentrosomal pole structure per se. Moreover, fluorescence recovery after photobleaching (FRAP) revealed that ZYG-9 turns over rapidly at acentrosomal poles, displaying similar turnover dynamics to tubulin itself, suggesting that ZYG-9 does not play a static structural role at poles. Together, these data support a global role for ZYG-9 in regulating the stability of bipolar spindles and demonstrate that the maintenance of acentrosomal poles requires factors beyond those acting to organize the pole structure itself

Metaphase-arrested spindles remain stable without auxin treatment.

Shows a metaphase-arrested oocyte expressing mCherry::tubulin (left) and degron::GFP::ZYG-9 (right), dissected into a control Meiosis medium solution. Corresponds to Fig 2A. No major changes in spindle length or shape occur. Note that the spindle rotates end-on for a portion of the video, but when it rotates back it is clear that the morphology of spindle has not changed. Time elapsed shown in (min):(sec). Scale bar = 5μm. (MOV)</p

FRAP of ASPM-1 at the poles of acentrosomal oocyte spindles.

Shows a Metaphase II oocyte spindle labeled with GFP::ASPM-1 and mCherry::histone; the polar body is not visible in the selected z-stacks. During the video the pole on the left is photobleached. Corresponds to Fig 7C. Time elapsed shown in seconds. Scale bar = 2.5μm. (MOV)</p

ASPM-1 marked monopole is not disrupted upon acute ZYG-9 AID depletion.

Shows a metaphase-arrested klp-18(RNAi) oocyte expressing GFP::ASPM-1, mCherry::tubulin (left) and degron::GFP::ZYG-9 (right) dissected into a Meiosis Medium solution containing 100μM auxin. Corresponds to Fig 6B. Although ZYG-9 was depleted from the oocyte, no disruption of monopole organization could be observed. Time elapsed shown in (min):(sec). Scale bar = 5μm. (MOV)</p

Monopolar spindles remain stable without auxin treatment.

Shows a metaphase-arrested klp-18(RNAi) oocyte expressing mCherry::tubulin (left) and degron::GFP::ZYG-9 (right) dissected into a control Meiosis Medium solution. Corresponds to Fig 6A. No major changes in monopole organization occurs during the course of the movie. Time elapsed shown in (min):(sec). Scale bar = 5μm. (MOV)</p

Model of acentrosomal pole coalescence and stability provided by ZYG-9.

(A) ZYG-9 is required to establish and maintain acentrosomal pole stability during meiosis. Removal of ZYG-9 prior to spindle assembly prevents multipolar spindles from stably coalescing into bipolar spindles. Additionally, removing ZYG-9 from stable bipolar spindles causes splaying of microtubule bundles near chromosomes and fragmentation of acentrosomal poles, ultimately causing the spindle to lose bipolarity and revert to a multipolar state. (B) Removal of ZYG-9 from a monopolar spindle causes no obvious defects to spindle morphology or monopole stability, suggesting that ZYG-9’s role in spindle bipolarity is not tied directly to acentrosomal poles. (C) Model: ZYG-9 is required for proper microtubule dynamics within the acentrosomal spindle, which is critical for the organization of tiled microtubules into stable bundles via microtubule motors/MAPs. Removal of ZYG-9 leads to defects in microtubule stabilization, leading to rapid splitting of acentrosomal poles, splaying of midspindle microtubule bundles, and loss of spindle bipolarity.</p

TAC-1 and ZYG-9 are interdependent for localization to acentrosomal poles.

(A) IF imaging of oocyte spindles; shown are DNA (blue), TAC-1 (green), ZYG-9 (stained with a GFP antibody; red), and tubulin (not shown in merge). Colocalization of TAC-1 and ZYG-9 is evident in metaphase and persists throughout anaphase (12/12 spindles). DNA and tubulin channels were deconvolved, while ZYG-9 and TAC-1 channels were not due to higher background staining of the TAC-1 antibody. Bars = 2.5μm. (B) IF imaging of oocyte spindles in the ZYG-9 AID strain in Metaphase I-arrest (emb-30(RNAi)) conditions; shown are DNA (blue), tubulin (green), TAC-1 (red), and ZYG-9 (stained with a GFP antibody; not shown in merge). Short-term ZYG-9 depletion via addition of auxin disrupts localization of TAC-1 to acentrosomal poles (8/8 spindles). As in (A), ZYG-9 and TAC-1 channels were not deconvolved. Bars = 2.5μm.</p