COPII, a coat protein complex that forms vesicles on the endoplasmic reticulum (ER), mediates trafficking to the Golgi. While metazoans have few genes encoding each COPII component, plants have expanded these gene families, leading to the hypothesis that plant COPII has functionally diversified. In the moss Physcomitrium (Physcomitrella) patens, the Sec23/24 gene families are each composed of seven genes. Silencing Sec23/24 revealed isoform-specific contributions to polarized growth, with the closely related Sec23D/E and Sec24C/D essential for protonemal development. Focusing on Sec23, we discovered that Sec23D/E mediate ER-to Golgi transport and are essential for tip growth, with Sec23D localizing to presumptive ER exit sites. By contrast, Sec23A,B,C,F, and G are dispensable and do not quantitatively affect ER-to-Golgi trafficking. However, Δsec23abcfg plants exhibited reduced secretion of plasma membrane cargo. Of the four highly expressed protonemal Sec23 genes, Sec23F/G are members of a divergent Sec23 clade specifically retained in land plants. Notably, Sec23G accumulates on ER-associated foci that are significantly larger, do not overlap with, and are independent of Sec23D. While Sec23D/E form ER exit sites and function as bona fide COPII components essential for tip-growing protonemata, Sec23G and the closely related Sec23F have likely functionally diversified, forming separate and independent ER exit sites and participating in Golgi-independent trafficking pathways.

30COPII, a coat of proteins that form vesicles on the ER, mediates vesicle 31 traffic from the ER to the Golgi. In contrast to metazoans that have few genes 32 encoding each COPII component, plants have expanded these gene families 33 leading to the hypothesis that plant COPII has functionally diversified. Here, we 34 analyzed the gene families encoding for the Sec23/24 heterodimer in the moss 35 Physcomitrium (Physcomitrella) patens. In P. patens, Sec23 and Sec24 gene 36 families are each comprised of seven genes. We discovered that Sec23D is 37 functionally redundant with Sec23E and together they are essential for 38 protonemal development. Sec23D is critical for secretion to the Golgi and the 39 plasma membrane in tip growing protonemata. Sec23D preferentially associates 40 with Sec24C/D, also essential for protonemata. In contrast, the remaining five 41

Exocytosis is a fundamental process used by all eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, we have named pHusion, for the analysis of exocytosis written in Image-Tank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are much longer lasting than those in immature murine cortical neurons, and that clustering of exocytic events occurs across cell types.

Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR-Cas9 mediated genome editing, P. patens was unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR-Cas9, molecular manipulations in P. patens have greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants.

No abstract

No abstract