A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at ER-PM contact sites

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1, 2, 3, 4, 5, 6, 7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis

Characterization and Functional Analysis of NET3C and VAP27-1 Interacting Proteins in Plants

In plants, the cortical ER network connects to the plasma membrane via the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins (e. g. NET3C, VAP27-1 and SYT1) and the cytoskeleton. However, little is known about the mechanism of regulation of the cytoskeleton at the plant EPCS, and its biological functions remain poorly understood. From previous protein-protein interaction screens, hundreds of candidate proteins that potentially interact with NET3C or VAP27-1 were identified, and three proteins, KLCR1, AtBRO1 and SINE2 were selected for further study. Firstly, microtubule binding proteins, KLCR1 and IQDs were identified to interact with NET3C and form an intermediate component of the plant EPCS. The NET3C-KLCR1-IQDs protein complex mediates links between actin filaments and microtubules, and their overexpression enhances their association. The expressions of NET3C and IQDs also have a direct influence to the ER morphology, with more ER polygonal and cisternal structures formed when their expression level is high. The loss of function of KLCR1 results in disorganization of both actin filaments and microtubules, which causes left handed helical growth of roots and defects in the shape of cotyledon pavement cells. AtBRO1 is another protein that has been confirmed to interact with NET3C. It localized to punctate structures and associated with the ER network and actin filaments in N. benthamiana leaf epidermal cells. Reverse-genetic analyses suggested the knockout AtBRO1 expression is lethal, and defective development was observed in about a quarter of seeds in siliques from heterozygous plants. Additionally, we also identified a protein complex which is comprised of VAP27-1, NET3A and SINE2. This protein complex links the nuclear envelope with the ER network and actin filaments and is likely to regulate the morphology of ER network and nuclear envelope during mitosis in plants. In summary, our results revealed novel NET3C and VAP27-1 complexes that are important for the ER morphology, cytoskeleton structures and plant development

Keep in contact: Multiple roles of endoplasmic reticulum-membrane contact sites and the organelle interaction network in plants

Functional regulation and structural maintenance of the different organelles in plants contribute directly to plant development, reproduction and stress responses. To ensure these activities take place effectively, cells have evolved an inter-connected network amongst various subcellular compartments, regulating rapid signal transduction and the exchange of biomaterial. Many proteins that regulate membrane connections have recently been identified in plants and this is the first step in elucidating both the mechanism and function of these connections. Amongst all organelles, the endoplasmic reticulum is the key structure which likely links most of the different subcellular compartments through membrane contact sites (MCS) and the ER-PM contact sites (EPCS) have been the most intensely studied in plants. However, the molecular composition and function of plant MCS are being found to be different from other eukaryotic systems. In this article, we will summarize the most recent advances in this field, and discuss the mechanism and biological relevance of these essential links in plants

Exo84c interacts with VAP27 to regulate exocytotic compartment degradation and stigma senescence

In plants, exocyst subunit isoforms exhibit significant functional diversity in that they are involved in either protein secretion or autophagy, both of which are essential for plant development and survival. Although the molecular basis of autophagy is widely reported, its contribution to plant reproduction is not very clear. Here, we have identified Exo84c, a higher plant-specific Exo84 isoform, as having a unique function in modulating exocytotic compartment degradation during stigmatic tissue senescence. This process is achieved through its interaction with the ER localised VAP27 proteins, which regulate the turnover of Exo84c through the autophagy pathway. VAP27 recruits Exo84c onto the ER membrane as well as numerous ER-derived autophagosomes that are labelled with ATG8. These Exo84c/exocyst and VAP27 positive structures are accumulated in the vacuole for degradation, and this process is partially perturbed in the exo84c knock-out mutants. Interestingly, the exo84c mutant showed a prolonged effective pollination period with higher seed sets, possibly because of the delayed stigmatic senescence when Exo84c regulated autophagy is blocked. In conclusion, our studies reveal a link between the exocyst complex and the ER network in regulating the degradation of exocytosis vesicles, a process that is essential for normal papilla cell senescence and flower receptivity

A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at Endoplasmic Reticulum-Plasma Membrane Contact Sites (EPCS)

In plants, the cortical ER network is connected to the plasma membrane through the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins and the cytoskeleton [1-7] . Strong co-alignment between EPCS and the cytoskeleton is observed in plants [1, 8], but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by FRET-FLIM analysis, to identify two microtubule binding proteins, KLCR1 (Kinesin Light Chain Related protein 1) and IQD2 (IQ67-Domain 2) that interact with the actin binding protein NET3C and form a component of plant EPCS, that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss of function mutants, net3a/NET3C RNAi, klcr1 or iqd2, exhibit defects in pavement cell morphology which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS, and for normal plant cell morphogenesis

Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery

The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1