Examining cell shape as the mechanism for furrow positioning in the early Zebrafish (Danio rerio) embryo

22 p.Through previous studies, it has been found that Zebrafish embryos show a predictable pattern of cell division consisting of the spindle apparatus always aligning perpendicular to its orientation in the previous cell stage. Although a predictable pattern for cell division has been determined, the exact mechanisms for the spindle positioning are unknown. One proposed determinant of spindle alignment is cell shape. This study aims to quantitatively analyze certain inherent characteristics of cells in early Zebrafish embryos to observe any relationships or trends that may occur. Patterns observed in these quantitative measurements may eventually help to formulate a model that predicts this pattern of cell division. The data shows a relationship between cell geometry and the orientation of the spindle axis by demonstrating that a greater difference between the long and short axis lengths and a ratio of these lengths further from 1.0 corresponds to a smaller angle difference between the spindle and furrow axis

Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility

Cytoplasmic dynein is a protein complex that transports molecular cargo along microtubules (MTs), playing a key role in the intracellular trafficking network. Vertebrate dynein's movement becomes strikingly enhanced upon interacting with dynactin and a cargo adaptor such as BicaudalD2. However, the mechanisms responsible for increased transport activity are not well understood, largely owing to limited structural information. We used cryo-electron tomography (cryo-ET) to visualize the 3D structure of the MT-bound dynein-dynactin complex from Mus musculus and show that the dynactin-cargo adaptor complex binds two dimeric dyneins. This configuration imposes spatial and conformational constraints on both dynein dimers, positioning the four motor domains in proximity to one another and oriented toward the MT minus end. We propose that grouping multiple dyneins onto a single dynactin scaffold promotes collective force production, increased processivity, and unidirectional movement, suggesting mechanistic parallels to axonemal dynein. These findings provide structural insights into a previously unknown mechanism for dynein regulation

Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction.

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis

Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting preferred orientations on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps

Defects in the vegetal localization of <i>wnt8a</i> mRNA and Sybu protein.

<p>A–D) Off-center shift of <i>wnt8a</i> mRNA is affected in <i>hec</i> mutants. Whole mount in situ hybridization of wild-type embryos (A,C) and <i>hec</i> mutant embryos (B,D) at the 1- (A,B, 30 mpf) and 4- (C,D, 60 mpf) cell stages. Images show representative embryos. A majority of wild-type embryos showed a clear off-center shift (85%, n = 27 at 30 mpf and 74%, n = 47 at 60 mpf). A majority of <i>hec</i> mutant embryos showed vegetal localization without a shift at 30 mpf (79%, n = 33, remaining embryos show no localization) and absence of localization at 60 mpf (89%, n = 38, remaining embryos show reduced vegetal localization without a shift). The apparent label at the base of the blastodisc is observed in a majority of mutant embryos (71%, n = 38) but not in wild-type (C) or control embryos labeled with other probes (not shown) and may reflect remaining <i>wnt8a</i> mRNA that has lost anchoring at the vegetal pole and has moved animally through the action of axial streamers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Fuentes1" target="_blank">[83]</a>. E–I) Localization of Sybu protein is affected in <i>hec</i> mutants. Whole mount immunofluorescence to detect Sybu protein of untreated wild-type (E,G) and <i>hec</i> mutant (F,H,) embryos and nocodazole-treated wild-type embryos (I) at the indicated stages. In wild-type embryos, an off-center shift in Sybu protein can be observed starting at 30 mpf (G). In <i>hec</i> mutants, Sybu protein becomes undetectable levels by this same time point (H). Patterns of localization of Sybu protein at 10 mpf and 20 mpf time points (combined n: 32 WT, 19 mutant for 10–20 mpf), and 30 mpf and 40 mpf time points were similar and have been combined. 59% (n = 32) of wild-type and 63% (n = 19) of <i>hec</i> mutant embryos showed centered vegetal localization during 10–20 mpf. At 30–40 mpf, the percent of embryos that showed vegetal localization, now with an off-center shift, was reduced to 25% (n = 28) in wild-type, and 0% (n = 25) of <i>hec</i> mutants showed any localization at these time points. Treatment of wild-type embryos with nocodazole inhibits the shift but does not result in delocalization from the vegetal cortex (I, embryo at 40 mpf), as previously shown <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>. Magnification bars in (D) and (I) correspond to 100 µm for panels sets (A–D) and (E–I), respectively.</p

Molecular identification of the <i>hecate</i> locus.

<p>A) Linkage map of the <i>hec</i> locus. The number of recombinants over the total number of analyzed meiosis is indicated. <i>hec</i> linkage was initially identified between SSLP markers z59658 and z24511 on chromosome 8. Fine mapping analysis with newly identified RFLP markers further narrowed the region between the gene <i>gpd1a-1</i> and the RFLP zC150E8y. B) Contig of five BAC clones covering the <i>hec</i> critical region. CH73-233M11, CH73-272M14, CH73-250D21, DKEY-43H14 and CH211-150E8 are five sequenced and overlapping BAC clones in this interval. C) Exon-intron structure of the <i>hec/grip2a</i> gene, which contains 16 exons. The <i>hec^p06ucal</i>, <i>hec^t2800</i> and <i>hec^p08ajug</i> alleles each cause a premature stop-codon in exon 4, exon 10 and exon 12, respectively. D) Sequence traces of the cDNA products from wild-type and the three mutant <i>hec</i> alleles. Nucleotide substitutions are indicated by the red box. Mutant cDNAs show a C-A transversion in codon 118 (<i>hec^p06ucal</i>), a C-T transversion in codon 414 (<i>hec^t2800</i>), or a C-T transversion in codon 499 (<i>hec^p08ajug</i>), all creating premature STOP codons. E) Schematic diagram showing the protein domain structures of Grip2a in the wild-type and mutant alleles. Red boxes represent conserved PDZ domains.</p

Localization of <i>grip2a</i> mRNA in wild-type and mutant oocytes.

<p>A–C) Whole mount in situ hybridization of dissected ovaries from wild-type (A), <i>bucky ball (buc</i>, B) and <i>magellan (mgn</i>, C) mutant females. In (A–C) oocytes at stage III of development are indicated. Smaller oocytes are at stages I and II, which are difficult to differentiate in whole mounts at this magnification. The <i>grip2a</i> mRNA localization domain is observed in an asymmetric cortical position in wild-type oocytes (A) but is unlocalized and diffuse in <i>buc</i> oocytes (B) and internally-located in <i>mgn</i> mutants (C). D–J) Sections of wild-type and mutant oocytes at the indicated stages after labeling to detect <i>grip2a</i> mRNA. Stages are as indicated in the panel and were determined by size and oocyte morphology according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Selman1" target="_blank">[101]</a>. D–F) Wild-type oocytes showing localization to the presumptive Balbiani Body (D) and subsequent localization to a cortical domain of the oocyte corresponding to the presumptive vegetal pole (E–F). G,H) <i>buc</i> mutant oocytes lack the Balbiani body <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Bontems1" target="_blank">[39]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Marlow1" target="_blank">[43]</a> and the <i>grip2a</i> mRNA subcellular localization domain in stage I and II oocytes. I) <i>mgn</i> mutant stage I oocytes exhibit an enlarged Balbiani body <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Gupta1" target="_blank">[40]</a> and displayed an enlarged <i>grip2a</i> mRNA localization domain. J) Stage II mgn mutant oocytes fail to localize transcripts to the vegetal pole which instead persist in an internal domain <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Gupta1" target="_blank">[40]</a>, as observed also for <i>grip2a</i> mRNA. Number of oocytes examined were as follows: wild-type: early stage I: 13, stage II: 12; <i>buc</i>: early stage I: 35, stage II: 26; <i>mgn</i>: early stage I: 23, stage II: 18. Magnification bar in (C) corresponds to 250 µm for panels (A–C), and in (J) to 50 µm for panels (D–J).</p

Amplification of <i>hecate/grip2a</i>-dependent symmetry breaking event by a general animal-directed long-range transport system.

<p>A) Cortical shifts of various vegetally localized components, including <i>wnt8a</i> mRNA, Sybu protein and <i>grip2a</i> mRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Lu1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Tran1" target="_blank">[8]</a>; this report) are short-range and dependent on microtubule bundling and alignment, itself dependent on <i>hec</i> function. In wild-type embryos, such a short-range shift generates a symmetry breaking event that is subsequently amplified by long-range, animally-directed transport mechanism independent of <i>hec</i> function and not restricted to the prospective dorsal axis. B) In <i>hec</i> mutant embryos, neither reorganization of vegetal microtubules into aligned bundles nor a short-range shift occur, so that, even though long-range transport remains intact, vegetal determinant transport to the animal pole is affected. The mechanistic basis for the long-range transport, occurring in the region of a loosely organized mediolateral microtubule cortical network remains to be determined (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#s3" target="_blank">Discussion</a>).</p