During plant cytokinesis membrane vesicles are efficiently delivered to the cell-division plane, where they fuse with one another to form a laterally expanding cell plate. These membrane vesicles were generally believed to originate from Golgi stacks. Recently, however, it was proposed that endocytosis contributes substantially to cell-plate formation. To determine the relative contributions of secretory and endocytic traffic to cytokinesis, we specifically inhibited either or both trafficking pathways in Arabidopsis. Blocking traffic to the division plane after the two pathways had converged at the trans-Golgi network disrupted cytokinesis and resulted in binucleate cells, whereas impairment of endocytosis alone did not interfere with cytokinesis. By contrast, inhibiting ER-Golgi traffic by eliminating the relevant BFA-resistant ARF-GEF caused retention of newly synthesized proteins, such as the cytokinesis-specific syntaxin KNOLLE in the ER, and prevented the formation of the partitioning membrane. Our results suggest that during plant cytokinesis, unlike animal cytokinesis, protein secretion is absolutely essential, whereas endocytosis is not.

Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, μ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their μ subunits in plants. In contrast, the μ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 μ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.adaptor complex 1 | membrane traffic | secretory pathway | development

The partitioning membrane of dividing plant cells is made by homotypic fusion of trans-Golgi network–derived membrane vesicles delivered to the division plane. The cytokinesis-specific syntaxin of Arabidopsis forms two different types of SNARE complexes, which can functionally replace each other in membrane fusion during cytokinesis.

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Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily -KNOLLE/SYP111, PEN1/SYP121 and SYP132 -in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion. SNARE proteins constitute a family of membraneanchored proteins that play key roles in membrane fusion events of intracellular trafficking pathways by forming SNARE complexes that dock membranes to be fused. Their main characteristic feature is an evolutionarily conserved domain of 60-70 amino acids arranged in heptad repeats, which has been designated the SNARE domain (1). Based on the conserved amino-acid residue at the center of the SNARE domain, SNARE proteins have been classified into R-(arginine) and Q-(glutamine) SNAREs. The Q-SNARE family is further divided into four subfamilies (Qa-, Qb-, Qc-and Qb,cSNAREs) based on differences in the structure of the SNARE domain (2). Each SNARE complex is formed by association of four interacting SNARE domains, one each from VAMP/R-SNARE on the donor membrane and three from Q-SNAREs on the acceptor membrane: one from syntaxin/Qa-SNARE and two from either SNAP25/Qb,c-SNARE or one each from two t-SNARE light chains/Qband Qc-SNAREs (1).

The TRIM-NHL protein Brain tumor (Brat) acts as a tumor suppressor in the brain, but how it suppresses tumor formation is not completely understood. Here, we combine temperature-controlled RNAi with transcriptome analysis to identify the immediate Brat targets in neuroblasts. Besides the known target Deadpan (Dpn), our experiments identify the transcription factor Zelda (Zld) as a critical target of Brat. Our data show that Zld is expressed in neuroblasts and required to allow re-expression of Dpn in transit-amplifying intermediate neural progenitors. Upon neuroblast division, Brat is enriched in one daughter cell where its NHL domain directly binds to specific motifs in the 3'UTR of and mRNA to mediate their degradation. In mutants, both Dpn and Zld continue to be expressed, but inhibition of either transcription factor prevents tumorigenesis. Our genetic and biochemical data indicate that Dpn inhibition requires higher Brat levels than Zld inhibition and suggest a model where stepwise post-transcriptional inhibition of distinct factors ensures sequential generation of fates in a stem cell lineage.

The COP9 signalosome (CSN) is required for the full activity of cullin-RING E3 ubiquitin ligases (CRLs) in eukaryotes. CSN exerts its function on CRLs by removing the ubiquitin-related NEDD8 conjugate from the cullin subunit of CRLs. CSN seems, thereby, to control CRL disassembly or CRL subunit stability. In Arabidopsis thaliana, loss of CSN function leads to constitutive photomorphogenic (cop) seedling development and a post-germination growth arrest. The underlying molecular cause of this growth arrest is currently unknown. Here, we show that Arabidopsis csn mutants are delayed in G2 phase progression. This cell cycle arrest correlates with the induction of the DNA damage response pathway and is suggestive of the activation of a DNA damage checkpoint. In support of this hypothesis, we detected gene conversion events in csn mutants that are indicative of DNA doublestrand breaks. DNA damage is also apparent in mutants of the NEDD8 conjugation pathway and in mutants of the E3 ligase subunits CULLIN4, COP1 and DET1, which share phenotypes with csn mutants. In summary, our data suggest that Arabidopsis csn mutants undergo DNA damage, which might be the cause of the delay in G2 cell cycle progression.KEY WORDS: COP9 signalosome, Cell cycle, DNA damage Development 135, 2013Development 135, -2022Development 135, (2008 (Miséra et al., 1994; Chory et al., 1989;Deng et al., 1991). COP1 is a RING-type E3 ubiquitin ligase that mediates the degradation of several positive photomorphogenesis regulators (Osterlund et al., 2000;Seo et al., 2003;Seo et al., 2004). The human COP1 ortholog (RFWD2) has been implicated in c-JUN degradation and in DNA damage response following irradiation Wertz et al., 2004). Human COP1 is inactivated in response to DNA damage by ATM-dependent phosphorylation, and the consequent stabilization of its degradation target p53 induces a G1 cell cycle arrest . The function of DET1 only became clear when it was recognized that it is a subunit of a CULLIN4 (CUL4)-containing CRL, designated DCX DET1COP1 or CUL4-DDB1 DET1COP1 , which also includes COP1 and the adaptor subunit DAMAGED DNA-BINDING PROTEIN1 (DDB1) (Benvenuto et al., 2002;Dornan et al., 2004;Wertz et al., 2004;Yanagawa et al., 2004;Chen et al., 2006). Although these findings suggest that COP1 and DET1 function together in a CUL4-containing CRL, the COP1 monomer alone has in vitro E3 ligase activity. It is therefore presently unclear which functions of COP1 require the E3 complex (and DET1) and which functions are mediated by COP1 alone.Arabidopsis csn mutants arrest growth at the seedling stage. The underlying molecular cause of this growth arrest remains to be identified. Here we show that csn mutant cells have a delay in G2 phase progression. This delay correlates with the activation of the DNA damage response pathway but is not exclusively induced by the DNA damage signaling kinases ATAXIA TELANGIECTASIA MUTATED (ATM) or WEE1. Our observation that gene conversion events can occur in csn mutants strongly argues that DNA doublestrand break...

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.