Pattabiraman, Divya et al. (2013) International Worm Meeting "Visualizing dynamics of meiotic prophase chromosome structures."

Chen, Grace, Pattabiraman, Divya, Villeneuve, Anne, Presler, Marc, Roelens, Baptiste

[International Worm Meeting, 2013]

The synaptonemal complex (SC) is a highly-ordered proteinaceous structure that assembles at the interface between aligned homologous chromosome pairs during meiotic prophase. Although EM images of SCs give the impression of a rigid, scaffold-like structure, recent studies suggest that the SC may be much more dynamic than previously appreciated. We are investigating the dynamics of the SC structure by using FRAP (Fluorescence Recovery After Photobleaching) to visualize the exchange of SC components within fully assembled SCs in pachytene nuclei of intact worms expressing a functional GFP-tagged version of SYP-3, a component of the SC central region. This approach has revealed a previously hidden dynamics of the SC, demonstrating that the SC is a malleable structure rather than a fixed scaffold. We observe half-maximal FRAP for GFP::SYP-3 by 10-15 minutes post-bleach, and recovery approaches a maximum by 1-1.5 h. Although this recovery time scale is slower than those observed for nucleoplasmic proteins (seconds) or microtubules in the first mitotic spindle (a few minutes), it is short relative to the length of the pachytene stage when full-length SCs are present (18-24 h). Thus, the observed dynamics indicate that the SC has the potential to undergo substantial remodeling and reorganization in response to different ongoing events of meiotic prophase. Based on our analyses, we can draw several conclusions: 1)Recovery occurs primarily by redistribution/exchange of SC subunits within the nucleus, and minimally by import of new SC subunits 2)The extent of SC dynamics decreases during pachytene progression and 3)SC dynamics characteristic of wild-type early pachytene were observed in mid and late pachytene nuclei in a zhp-3 mutant, wherein chromatin and nuclear envelope features that are normally limited to early pachytene are prolonged. Together our data suggest that the dynamic state of the SC structure is an actively regulated feature of the meiotic program.

Pattabiraman, Divya et al. (2015) International Worm Meeting "Meiotic Progression and Recombination Modulate the Dynamic State of the Synaptonemal Complex."

Pattabiraman, Divya, Roelens, Baptiste, Presler, Marc, Villeneuve, Anne, Chen, Grace

[International Worm Meeting, 2015]

The synaptonemal complex (SC) is a highly-ordered proteinaceous structure that assembles at the interface between aligned homologous chromosome pairs during meiotic prophase. The SC is integral to most aspects of the meiotic program, with demonstrated roles in stabilization of homolog pairing, in promoting the formation of crossovers, in enabling meiotic progression and in crossover interference. Although EM images of SCs give the impression of a rigid, scaffold-like structure, recent studies suggest that the SC may be much more dynamic than previously appreciated. We have investigated the dynamics of the SC structure by using FRAP (Fluorescence Recovery After Photobleaching) analysis to visualize the exchange/turnover of SC components within fully assembled SCs. This was done in pachytene nuclei of intact worms expressing a GFP-tagged version of SYP-3, a component of the SC central region.This approach has revealed a previously hidden dynamics of the SC, demonstrating that the SC is a malleable structure rather than a fixed scaffold. We observe significant recovery of GFP::SYP-3 by 10-15 minutes post-bleach, and recovery approaches a maximum by 1-1.5 h. Although this recovery time scale is slower than those observed for nucleoplasmic proteins (seconds) or spindle microtubules in the first mitotic spindle (a few minutes), it is short relative to the length of the pachytene stage when full-length SCs are present (18-24 h). Thus, the observed dynamics indicate that the SC has the potential to undergo substantial remodeling and reorganization in response to ongoing events of meiotic prophase. Based on our analyses, we can draw several conclusions: 1) Recovery occurs primarily by redistribution/exchange of existing SC subunits within the nucleus, and minimally by import of new SC subunits; 2) During wild-type meiosis, the extent of SC dynamics decreases during normal progression through the pachytene stage; and 3) In crossover defective mutants (spo-11, zhp-3 and cosa-1), the more dynamic state of the SC characteristic of early pachytene, is prolonged, suggesting a role for recombination in regulating SC dynamics. These conclusions have been corroborated by experiments conducted using two very different imaging systems and two different approaches for data analysis. Together our findings indicate that the dynamic state of the SC structure is an actively regulated feature of the meiotic program. Moreover, we propose that SC dynamics is likely modulated by the same network that regulates meiotic progression in response to monitoring the status of recombination intermediates.

Divya Pattabiraman et al. (2010) East Asia Worm Meeting "Wnt signals and Frizzled receptors regulate dendrite formation in C. elegans"

Divya Pattabiraman, Massimo A. Hilliard, Brent Neumann, Leonie Kirszenblat

[East Asia Worm Meeting, 2010]

Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. While many molecules involved in axon development have been discovered, there is little known about the ligands and receptors that regulate dendrite development. To understand how dendrites develop in C. elegans we focused on the PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the gcy-36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in lin-17, a C. elegans Frizzled receptor gene. We found that in lin-17(vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a non-cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that lin-44 mutants presented PQR dendrite defects similar to those observed in lin-17 mutants. We expressed LIN-44 ectopically from more anterior regions of the body and found that it rescued the PQR dendrite defects of lin-44 mutants, indicating that LIN-44 functions as a permissive cue. Using a heat-shock promoter to drive LIN-44 we determined that the presence of this ligand at the time of PQR dendrite formation was sufficient to rescue the dendrite defects of the lin-44 mutant. Analysis of the lin-17 lin-44 double mutant indicated a genetic interaction between these molecules. Our studies provide the first direct evidence that specific Wnt signals and Frizzled receptors regulate dendrite formation in vivo. We propose a model in which PQR dendrite formation is achieved by the interaction of LIN-44 and LIN-17 acting on PQR through its neighbouring cells.

Kirszenblat, Leonie et al. (2011) International Worm Meeting "Wnt signals and Frizzled receptors regulate dendrite formation in C. elegans ."

Pattabiraman, Divya, Hilliard, Massimo A., Neumann, Brent, Kirszenblat, Leonie

[International Worm Meeting, 2011]

Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. While many molecules involved in axon development have been discovered, there is little known about the ligands and receptors that regulate dendrite development. To understand how dendrites develop in C. elegans we focused on the PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the gcy- 36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in lin-17, a C. elegans Frizzled receptor gene. We found that in lin17(vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that lin-44 mutants presented PQR dendrite defects similar to those observed in lin-17 mutants. We expressed LIN-44 ectopically from more anterior regions of the body and found that it worsened the PQR dendrite defects of lin-44 mutants, indicating LIN-44 functions as an instructive cue. Furthermore, we induced LIN-44 expression at different stages of development and found that expression of this molecule is necessary and sufficient prior to PQR dendrite formation. Analysis of the lin-17 lin-44 double mutant indicated a genetic interaction between these molecules. Our studies provide the first direct evidence that specific Wnt signals and Frizzled receptors regulate dendrite formation in vivo. We propose a model in which LIN-17, present on the cell surface of PQR, interacts with LIN-44 through an attractive mechanism to mediate dendrite formation.

Kirszenblat, Leonie et al. (2009) International Worm Meeting "Wnt and Frizzled molecules regulate dendrite formation in C. elegans. ."

Neumann, Brent, Kirszenblat, Leonie, Pattabiraman, Divya, Hilliard, Massimo A.

[International Worm Meeting, 2009]

Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. While many molecules involved in axon development have been discovered, there is little known about the molecules and mechanisms required for dendrite formation. To understand how dendrites develop in C. elegans we have chosen to study the oxygen sensory neuron, PQR. The PQR neuron has its cell body positioned in the left lumbar ganglion on the posterior-lateral body. From the cell body two processes extend. A single dendrite extends posteriorly towards the tail ending with a cilium in the pseudocoelom. On the opposite side, an axon extends anteriorly joining the ventral nerve cord and terminating in the midbody region. PQR is born post-embryonically during the L1 stage, allowing easy visualization of dendrite and axon development using the gcy-36::GFP transgene. We have used a candidate-mutant approach to identify genes involved in dendrite formation. The secreted Wnt ligand, LIN-44, regulates neuronal polarity in PLM mechanosenosory neurons in the C. elegans tail. We found that LIN-44 specifically regulates dendrite formation in PQR. In lin-44 mutants, the PQR dendrite was often shortened or absent. This defect appeared in the initial stages of development (at the L1 stage) and persisted until adulthood, suggesting that the phenotype results from a failure in dendrite development rather than the presence of ectopic pruning. Interestingly, the PQR axon was not affected in these animals, indicating a dendrite-specific effect of LIN-44 on this cell. LIN-44 is expressed by hypodermal cells in the tip of the tail, a position that is posterior to the PQR cell body. The PQR dendrite develops towards the region where LIN-44 is expressed, and thus this ligand may act as an attractant cue for the growing dendrite. LIN-17/Frizzled acts as a receptor for LIN-44 in regulating neuronal polarity and other biological processes. We found that lin-17 mutant animals presented defects in PQR dendrite development similar to those observed in lin-44 mutants: PQR dendrite was shortened or absent, while the axon was unaffected. Thus, as for lin-44, the lin-17 gene has a dendrite specific effect. Our results suggest that Wnt molecules and Frizzled receptors are key molecules required for PQR dendrite formation.

Roelens, Baptiste et al. (2019) International Worm Meeting "The HAL-2/HAL-3 complex controls homolog pairing and synapsis by regulating polo-like kinase activity during C. elegans meiosis."

Pattabiraman, Divya, Girard, Chloe, Cutillas, Pedro, Woglar, Alex, Martinez-Perez, Enrique, Roelens, Baptiste, Barroso, Consuelo, Villeneuve, Anne, Montoya, Alex

[International Worm Meeting, 2019]

Segregation of homologous chromosomes is the landmark event of meiosis. This essential feature relies on the coordinated execution of a complex set of interconnected events: homologous chromosomes must first locate, recognize and align with their correct pairing partners during a period of active chromosome movement. This alignment is stabilized through the assembly of a meiosis-specific structure known as the synaptonemal complex (SC), which promotes formation of crossover recombination events between the DNA molecules of the homologs to create the linkages that enable their segregation. Here, we identify HAL-3 (Homolog Alignment 3), as a partner of the nucleoplasmic protein HAL-2 previously described as an important regulator of early prophase events in the model organism C. elegans (Zhang et al., 2012). We show that loss of hal-3 function phenocopies multiple defects observed in hal-2 mutant meiocytes, including failure to establish homolog pairing and inappropriate loading of SC subunits onto unpaired chromosome axes. Proper coordingation between homolog pairing and synapsis is ensured, in part, by the activity of the polo-like kinases PLK-1 and PLK-2, which dynamically localize at defined subnuclear locations during wild-type prophase progression. We show that loss of hal function results in misregulation of the subcellular localization of PLK-1 and PLK-2, and we provide evidence for PLK activity at ectopic locations in hal-2 and hal-3 mutants. Further, we find that loss of PLK-2 activity partially suppresses several meiotic defects in hal mutants, suggesting that the defects result from inappropriate PLK activity. Together our data support a model in which the nucleoplasmic HAL-2/HAL-3 complex regulates early meiotic prophase events by constraining both localization and activity of meiotic Polo-like kinases, both to prevent interaction with untimely targets and to promote proper association with stage-appropriate targets.