To test whether Pod-1 can crosslink actin and MTs, soluble Pod-1 was purified from a stable S2 cell line engineered to inducibly express full-length His-tagged Pod-1. When purified Dpod-1HIS (80-100 nM) is added to fluorescently labeled phalloidin-stabilized actin filaments (30-60 nM), it rapidly (within minutes) induces the formation of long (20-50 μm), mostly unbranched actin bundles that frequently have bends or curls. Their appearance does not change over time, suggesting that a steady state is reached. When buffer alone or BSA (even at a 25-fold higher concentration) is added to the actin filaments, no bundling activity is observed, demonstrating that the activity is specific. Similarly, when taxol-stabilized fluorescent MTs are combined with purified Pod-1HIS (80-100 nM Pod-1HIS; 500 nM MTs), rapid crosslinking of MTs is observed, whereas no crosslinking occurs with buffer alone or with BSA -- even at a 25-fold higher concentration. Thus, Pod-1HIS possesses both actin and MT crosslinking activities (Rothenberg, 2003).
To test whether Pod-1 can remodel the cytoskeleton in cells, Pod-1 was studied in S2 cells, a system that can be used to study cytoskeletal dynamics. In S2 cells plated and spreading on concanavalinA-coated (conA) cover slips, endogenous Pod-1 localizes to sites of new actin polymerization, particularly at the lamellar edge. Costaining fixed cells with Pod-1 and Alexa488-phalloidin shows that Pod-1 is enriched at the edge of ruffling lamellae in an uneven, punctate pattern. Notably, Pod-1 colocalizes with a subset of actin, particularly the newly polymerized actin assembled at the lamellar edge that supports retrograde flow. Prominent staining is also seen on intralamellar actin filaments and filopodia-like structures (Rothenberg, 2003).
Costaining spreading cells for Pod-1 and Tubulin shows Pod-1 colocalizing with a subset of MTs -- especially those whose polymerizing ends are meeting the lamellar edge. In the rare cells that project filopodia, Pod-1 accumulates to high levels in those projections (comprised of actin bundles) and colocalizes with invading MTs. This subcellular localization is consistent with a molecule playing a role in actin/MT interactions (Rothenberg, 2003).
To investigate the dependence of Pod-1's subcellular localization on actin and MTs, cells were treated with latrunculin, a drug that leads to the depolymerization of actin microfilaments, or nocodazole, a drug that causes depolymerization of MTs. Nocodazole does not change the subcellular localization of Pod-1 even when MTs are completely depolymerized; thus, Pod-1 localization is independent of MTs. In contrast, latrunculin disrupts the actin cytoskeleton and causes Pod-1 to lose its characteristic localization at the lamellar edge. Costaining for Pod-1 and Tubulin shows that in latrunculin-treated cells, Pod-1 relocalizes from actin filaments to MTs, suggesting that Pod-1 had a high affinity for actin and a lower affinity for MTs or that its association with MTs may be regulated. This finding, together with the endogenous Pod-1 localization and the observed biochemical activities, is consistent with a role for Pod-1 in the interaction of actin and MTs in dynamic cellular structures (Rothenberg, 2003).
When Pod-1HIS (80-100 nM) is added simultaneously to phalloidin-stabilized fluorescent actin filaments (30-60 nM) and taxol-stabilized fluorescent MTs (500 nM), a dramatic crosslinking activity is observed in which actin bundles colocalize with MT bundles. Significantly, when the same experiment is performed with purified α-actinin, a well-characterized actin bundling protein, actin aggregates are seen, but there is no bundling of MTs. Thus, in vitro, Pod-1 could crosslink actin filaments and MTs, activities that are likely to be significant for the remodeling and coordination of actin and MT networks in dynamic cells (Rothenberg, 2003).
Using several cDNA antisense probes derived from different regions of the pod-1 cDNA, in situ hybridization on 0-16 hr embryos was conducted to determine the mRNA expression pattern of pod-1 during embryogenesis. Cellularizing (stage 5) embryos demonstrate a ubiquitous maternal contribution of pod-1 mRNA. Embryos in early neurogenesis (stage 9) show high pod-1 expression in neuroblasts and in their progeny and low expression in the epidermis. Later on (stage 12), expression is seen in the developing PNS as well as the CNS. This pattern of high neuronal and low epidermal expression persists throughout embryogenesis. Western blot analysis with an anti-Pod-1 antibody on a 0-16 hr embryonic extract shows a single 130 kDa Pod-1 band, indicating that a single form of Pod-1 is expressed during embryogenesis (Rothenberg, 2003).
To determine the localization of Pod-1 in vivo, embryos were stained for Pod-1. At stage 12, during neurite growth in the CNS, Pod-1 staining is found at high levels in nascent axons. By stage 16/17, Pod-1 staining is abundant on all axons in the ventral nerve cord (VNC). Double labeling with anti-Pod-1 and 1D4/FasII shows Pod-1 staining in axon growth cones and along motor axons. Interestingly, Pod-1 is especially abundant at important navigational choice points, locations of axon turning or branching where growth cones slow down, enlarge, search the environment for guidance cues, and display precise coordination of actin and MTs. Although Pod-1 is expressed in all motorneurons, this is particularly noticeable in the ISN and in ISNb. ISN extends from the ventral nerve cord (VNC) to the dorsal musculature where it ramifies axons at three distinct choice points; at stage 16/17, Pod-1 concentrates at those points in each ISN in all embryos observed. Careful observation shows that Pod-1 appears to be in a subset of ISN growth cone processes at this stage. ISNb extends from the VNC into the ventral musculature where axons defasciculate and turn at three distinct choice points to innervate muscles 7, 6, 13, and 12. Pod-1 concentrates at the tips and choice points of ISNb axons as well in all embryos observed (Rothenberg, 2003).
RNAi was used to ask whether depletion of Pod-1 from S2 cells can alter cytoskeletal regulation or dynamics. Although RNAi treatment reduces Pod-1 to undetectable levels (by both immunostaining and Western blot), no changes were observed in cell shape, actin appearance, or localization, or MT appearance or localization in fixed cells. Similarly, when Pod-1-RNAi cells are transfected with ActinGFP or TubulinGFP to assay cytoskeletal dynamics by live imaging, no differences are observed from normal S2 cells. Parameters measured included rate and extent of retrograde actin flow, rate of MT growth and shrinkage, rate of MT catastrophe (transitions from growth or pause to shrinkage per second), rate of MT rescue (transitions from pause or shrinkage to growth per second), and proportions of paused, growing, and shrinking MTs. Thus, Pod-1 does not appear to be necessary for cytoskeletal regulation in S2 cells (Rothenberg, 2003).
Next, it was asked whether Pod-1 overexpression is sufficient to affect cytoskeletal networks. Overexpression of Pod-1GFP causes a dramatic dose-dependent remodeling of cell shape. Cells expressing high levels of Pod-1GFP (as determined by fluorescence intensity) extend long, neurite-like projections that are sometimes branched and are highly dynamic, displaying behaviors such as growth and extension, lateral movement along the cell surface, retrograde flow, and catastrophic collapse and retraction. These projections are even observed in cells growing in their culture dish before plating onto conA, indicating that their formation does not require the spreading signal provided by conA. In contrast, cells that are untransfected, transfected with ActinGFP, or expressing lower levels of Pod-1GFP are invariably discoid and do not display this dramatic behavior (Rothenberg, 2003).
Staining with rhodamine-phalloidin indicates that the processes not only contain high levels of Pod-1GFP but are also rich in actin bundles. To investigate whether these processes are actin dependent, live, plated cells were treated with latrunculin; the processes did not form. Similarly, treatment with latrunculin before plating on conA also blocked process formation. Taken together with the observation that Pod-1 localization to the lamellar edge in untransfected cells depends on actin, this shows that the processes are actin dependent (Rothenberg, 2003).
When fixed and stained for tubulin, many of the processes induced by Pod-1GFP are found to contain invading MTs, much like stabilized filopodia in neuronal growth cones. However, the processes are not dependent on MTs, since nocodazole treatment does not block their formation. Consistent with this result, nearly all the processes extend beyond the ends of invading MTs; thus, MT polymerization does not drive the extension of these processes (Rothenberg, 2003).
Also resembling the filopodia of axonal growth cones, the tips of a subset of the processes (presumably those that are growing) exhibit a focus of Enabled (Ena) expression; Ena is a cytoskeletal regulator that facilitates continued actin polymerization at the barbed ends of actin filaments, induces cellular projections when overexpressed, and functions together with several different receptors (including Robo and UNC-40/DCC) implicated in axon guidance (Rothenberg, 2003).
To determine whether Pod-1 has a specific role in axon guidance, the mutant phenotype of embryos lacking pod-1 was characterized. pod-1 is located at 6D1-2 on the X chromosome, a region containing no available deficiencies. Therefore, several null alleles of pod-1 were generated by imprecise excision of two nearby P elements. Four lethal deletions were identified that remove the entire coding sequence of pod-1: Δ17, Δ96, Δ225, and Δ291. This was confirmed by staining for Pod-1 in mutant embryos and mutant mitotic clones. Δ96 was chosen for further study since the only other gene besides pod-1 that the deletion apparently removed was CG4536, a putative TRP channel homologous to C. elegans osm-9 that is not expressed during axonogenesis (Rothenberg, 2003).
In zygotic mutants, although staining with BP102 (an antibody that reveals a regular ladder-like pattern of longitudinal and commissural VNC axons in wild-type embryos) yielded a relatively normal pattern, a low but significant frequency of axon defects was revealed in the motorneurons by 1D4/FasII staining. dsRNAi confirmed that the phenotype was in fact due to pod-1. These defects are most likely due to a primary defect in axon targeting, since no defects were found in neuroblast polarity (determined by examining the asymmetric localization of Bazooka, aPKC, Inscuteable, Miranda, Pon, Prospero, and Numb), mitotic spindle orientation (determined by β-tubulin staining), cell fate determination (determined by Even-skipped staining), or epidermal integrity (determined by Bazooka, DmPar6, aPKC, Armadillo, and Crumbs staining as well as by cuticle analysis of first instar larvae). Nonneuronal features of these embryos were also normal, including segmentation and muscle pattern (Rothenberg, 2003).
Embryos lacking all Pod-1 display a range of abnormalities in their VNC axons as revealed by BP102 or 1D4/FasII: thinning of longitudinals, abnormal midline crossing and wandering trajectories, axon tangles, axon breaks, collapse or thinning of the anterior and posterior commissurals, and defasciculation. All embryos devoid of Pod-1 stain with 1D4/FasII displayed defects, indicating that the phenotype is fully penetrant. These defects are significantly rescued when these embryos are induced to express Pod-1GFP by elavGal4, a postmitotic neural-specific driver, demonstrating that the phenotype is due to the absence of Pod-1 in differentiating neurons and that Pod-1GFP functions like Pod-1 (Rothenberg, 2003).
To more precisely determine the axonal phenotype by studying isolated nerves, motorneuron projections were assayed by 1D4 staining of embryos lacking all Pod-1. This analysis reveals frequent guidance defects in all the motorneuron projections that normally target body wall muscles: ISN, SNa, ISNb, SNc, and ISNd. Invariably, in mutants, ISN axons extend out from the VNC to the region of the first choice point, a distance of many cell diameters. However, in that region, ISN often displays defects such as stalling/splaying, defasciculation, and failure to innervate targets. This suggests that Pod-1 is required for the guidance of ISN growth cones approaching their targets but is not an essential factor in early axon outgrowth and extension. Normally, SNa defasciculates from the segmental nerve (SN) and projects dorsally in a tight fascicle until reaching the dorsal edge of muscle 12, a choice point where it defasciculates to form a dorsal and a lateral branch. In embryos lacking all Pod-1, SNa frequently exhibits defects: a missing or truncated dorsal or lateral branch, arrest/splaying at the choice point, extra branching, or abnormal defasciculation. Similarly, ISNb axons often display various abnormalities, such as failure to innervate the clefts between muscles 6/7 and 12/13, premature arrest (usually around muscle 13), failure to defasciculate from ISN, and bypass of targets. These ISN, SNa, and ISNb phenotypes are significantly rescued by postmitotic neural expression of Pod-1GFP (Rothenberg, 2003).
In summary, axons devoid of Pod-1 frequently demonstrate aberrant guidance with subsequent failure of target innervation, showing that Pod-1 is required for the fidelity of axon turning, branching, or extension past choice points. Importantly, no general problem with early axon outgrowth or extension out to navigational choice points is observed. Moreover, growth cone structure is not obviously disrupted, since filopodia are still seen. Thus, Pod-1 is not required for filopodia formation in axonal growth cones, an important point since growth cone filopodia are required for steering but not extension of axons. In addition, expressivity is variable: some nerves reached their targets even without any Pod-1. Thus, while dispensible for the early steps of axon elongation, Pod-1 is required for the fidelity of axon targeting (Rothenberg, 2003).
Since Pod-1 is necessary for the fidelity of axon targeting and can remodel the cytoskeleton in S2 cell, whether an overabundance of Pod-1 can disrupt axon targeting was tested. Using two copies each of elavGal4 and UAS-Pod-1GFP, Pod-1GFP was overexpressed postmitotically in neurons and multiple, severe axon guidance defects were observed in all axons examined. Longitudinal tracts in the VNC exhibit uneven fascicle shapes, abnormal trajectories, axon breaks, and fascicle collapse. ISN exhibits defasciculation, overbranching, and choice point abnormalities. SNa often shows missing or misplaced branches, abnormal defasciculation, and defective trajectories. ISNb frequently arrests and fails to innervate targets in the ventral musculature, remains fasciculated with ISN, or takes abnormal trajectories. These defects are all consistent with cytoskeletal abnormalities in growth cones leading to defective steering, branching, or extension (Rothenberg, 2003).
Interestingly, staining of these embryos with 1D4 (even using rapid fixation to preserve filopodia) did not reveal an obvious difference in the number or shape of filopodia, suggesting that Pod-1 overexpression has a more subtle effect on cytoskeletal networks in the growth cones than in the S2 cells (Rothenberg, 2003).
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date revised: 5 September 2003
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