Biology subjects

Bezanilla, M.

Publications and source records attributed to Bezanilla, M..

3 recordsLinked to original sources

bioRxiv2020

Regulation of Vacuole Morphology by PIEZO Channels in Spreading Earth Moss

The perception of mechanical force is a fundamental property of most, if not all cells. PIEZO channels are plasma membrane-embedded mechanosensitive calcium channels that play diverse and essential roles in mechanobiological processes in animals1,2. PIEZO channel homologs are found in plants3,4, but their role(s) in the green lineage are almost completely unknown. Plants and animals diverged approximately 1.5 billion years ago, independently evolved multicellularity, and have vastly different cellular mechanics5. Here, we investigate PIEZO channel function in the moss Physcomitrium patens, a representative of one of the first land plant lineages. PpPIEZO1 and PpPIEZO2 were redundantly required for normal growth, size, and shape of tip-growing caulonema cells. Both were localized to vacuolar membranes and facilitated the release of calcium into the cytosol in response to hypoosmotic shock. Loss-of-function ({Delta}Pppiezo1/2) and gain-of-function (PpPIEZO2-R2508K and -R2508H) mutants revealed a role for moss PIEZO homologs in regulating vacuole morphology. Our work here shows that plant and animal PIEZO homologs have diverged in both subcellular localization and in function, likely co-opted to serve different needs in each lineage. The plant homologs of PIEZO channels thus provide a compelling lens through which to study plant mechanobiology and the evolution of mechanoperceptive strategies in multicellular eukaryotes.

plant biology↗

bioRxiv2020

The COPII components Sec23 and Sec24 form isoform specific subcomplexes with Sec23D/E and Sec24C/D essential for tip growth

COPII, a coat of proteins that form vesicles on the ER, mediates vesicle traffic from the ER to the Golgi. In contrast to metazoans that have few genes encoding each COPII component, plants have expanded these gene families leading to the hypothesis that plant COPII has functionally diversified. Here, we analyzed the gene families encoding for the Sec23/24 heterodimer in the moss Physcomitrium (Physcomitrella) patens. In P. patens, Sec23 and Sec24 gene families are each comprised of seven genes. Silencing the Sec23/24 genes revealed isoform specific contributions to polarized growth, with the closely related Sec23D/E and Sec24C/D essential for protonemal development. Focusing on the Sec23 gene family, we discovered that loss of Sec23D alters ER morphology, increases ER stress, inhibits trafficking to the Golgi and to the plasma membrane in tip growing protonemata. In contrast, the remaining five Sec23 genes are dispensable for tip growth. While Sec23A/B/C/F/G do not quantitatively affect ER to Golgi trafficking in protonemata, they do contribute to secretion to the plasma membrane. Of the three highly expressed Sec23 isoforms in protonemata, Sec23G forms ER exit sites that are larger than Sec23B and Sec23D and do not overlap with Sec23D. Furthermore, ER exit sites labeled by Sec23B or Sec23G form in the absence of Sec23D. These data suggest that Sec23D/E form unique ER exit sites contributing to secretion that is essential for tip growing protonemata.

cell biology↗

bioRxiv2019

Efficient and Modular CRISPR-Cas9 Vector System for Physcomitrella patens

CRISPR-Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA-guided nuclease to initiate double-strand breaks. Until recently, classical RAD51-mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR-Cas9 mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Collonnier et al. 2017). CRISPR-Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR-Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR-Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology-directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5 and 3 ends of gene coding regions. With regards to homology-directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a \"stop codon cassette\" with down-stream near-effortless genotyping.

molecular biology↗