Byung-Ho Kang

Electron Tomography of RabA4b‐ and PI‐4Kβ1‐Labeled Trans Golgi Network Compartments in Arabidopsis

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin‐coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi‐associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30–35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi‐associated and free TGN compartments, but not trans Golgi cisternae, bind anti‐RabA4b and anti‐phosphatidylinositol‐4 kinase (PI‐4K) antibodies. RabA4b and PI‐4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA‐a1‐green fluorescent protein (GFP) and SYP61‐cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI‐4Kβ1/2 regulates SV size.

Nanoscale Architecture of Endoplasmic Reticulum Export Sites and of Golgi Membranes as Determined by Electron Tomography

Modern architecture is guided by the axiom “form follows function,” which emphasizes the need for the shape of a building or an object to reflect its intended function or purpose. Biologists tend to prefer the phrase “form begets function,” because in living organisms, form not only reflects

Caenorhabditis elegans drp-1 and fis-2 Regulate Distinct Cell-Death Execution Pathways Downstream of ced-3 and Independent of ced-9

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1s proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.

Identification and characterization of COPIa- and COPIb-type vesicle classes associated with plant and algal Golgi

Coat protein I (COPI) vesicles arise from Golgi cisternae and mediate the recycling of proteins from the Golgi back to the endoplasmic reticulum (ER) and the transport of Golgi resident proteins between cisternae. In vitro studies have produced evidence for two distinct types of COPI vesicles, but the in vivo sites of operation of these vesicles remain to be established. We have used a combination of electron tomography and immunolabeling techniques to examine Golgi stacks and associated vesicles in the cells of the scale-producing alga Scherffelia dubia and Arabidopsis preserved by high-pressure freezing/freeze-substitution methods. Five structurally distinct types of vesicles were distinguished. In Arabidopsis, COPI and COPII vesicle coat proteins as well as vesicle cargo molecules (mannosidase I and sialyltransferase–yellow fluorescent protein) were identified by immunogold labeling. In both organisms, the COPI-type vesicles were further characterized by a combination of six structural criteria: coat architecture, coat thickness, membrane structure, cargo staining, cisternal origin, and spatial distribution. Using this multiparameter structural approach, we can distinguish two types of COPI vesicles, COPIa and COPIb. COPIa vesicles bud exclusively from cis cisternae and occupy the space between cis cisternae and ER export sites, whereas the COPIb vesicles bud exclusively from medial- and trans-Golgi cisternae and are confined to the space around these latter cisternae. We conclude that COPIa vesicle-mediated recycling to the ER occurs only from cis cisternae, that retrograde transport of Golgi resident proteins by COPIb vesicles is limited to medial and trans cisternae, and that diffusion of periGolgi vesicles is restricted.

Auxin-Mediated Ribosomal Biogenesis Regulates Vacuolar Trafficking in Arabidopsis

This study describes the function of RPL4A, a ribosomal protein. It finds a link between ribosomal biogenesis and vacuolar protein sorting and provides insights into the auxin-mediated regulation of vacuolar trafficking in metabolically active tissues. In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis.

Miniature1-Encoded Cell Wall Invertase Is Essential for Assembly and Function of Wall-in-Growth in the Maize Endosperm Transfer Cell

The miniature1 (mn1) seed phenotype in maize (Zea mays) is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase (INCW2) that localizes exclusively to the basal endosperm transfer cells (BETCs) of developing seeds. A common feature of all transfer cells is the labyrinth-like wall-in-growth (WIG) that increases the plasma membrane area, thereby enhancing transport capacity in these cells. To better understand WIG formation and roles of INCW2 in the BETC development, we examined wild-type and mn1 mutant developing kernels by cryofixation and electron microscopy. In Mn1 seeds, WIGs developed uniformly in the BETC layer during 7 to 17 d after pollination, and the secretory/endocytic organelles proliferated in the BETCs. Mitochondria accumulated in the vicinity of WIGs, suggesting a functional link between them. In the mn1 BETCs, WIGs were stunted and their endoplasmic reticulum was swollen; Golgi density in the mutant BETCs was 51% of the Mn1 Golgi density. However, the polarized distribution of mitochondria was not affected. INCW2-specific immunogold particles were detected in WIGs, the endoplasmic reticulum, Golgi stacks, and the trans-Golgi network in the Mn1 BETCs, while immunogold particles were extremely rare in the mutant BETCs. Levels of WIG development in the empty pericarp4 mutant was heterogeneous among BETCs, and INCW2 immunogold particles were approximately four times more abundant in the larger WIGs than in the stunted WIGs. These results indicate that polarized secretion is activated during WIG formation and that INCW2 is required for normal development of WIGs to which INCW2 is localized.

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.

Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization

Eliminating paternal mitochondria During fertilization, the oocyte and sperm each bring their mitochondria to the union. Shortly afterward, the paternal mitochondria are degraded, and only the maternal mitochondria are conveyed to the progeny. Zhou et al. observed that the integrity of the inner membrane of paternal mitochondria is compromised, which apparently marks them for degradation (see the Perspective by van der Bliek). Autophagy commences by mitochondrial endonuclease G relocating from the intermembrane space into the matrix and subsequently degrading the paternal mitochondrial DNA. Any delay in this process increases embryonic lethality. Science, this issue p. 394; see also p. 351 A mitochondrial enzyme promotes the destruction and removal of sperm-derived mitochondria in nematode worm embryos. Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

CIS-GOLGI CISTERNAL ASSEMBLY AND BIOSYNTHETIC ACTIVATION OCCUR SEQUENTIALLY IN PLANTS AND ALGAE

The cisternal progression/maturation model of Golgi trafficking predicts that cis‐Golgi cisternae are formed de novo on the cis‐side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high‐pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus flytrap) as determined by electron microscopy, electron tomography and immuno‐electron microscopy techniques. Our findings are as follows: (i) The cis‐most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3–5 COPII vesicles in contact with a C2 cis cisterna. (ii) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (iii) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (iv) C2‐Cn cis cisternae grow through COPII vesicle fusion. (v) ER‐resident proteins are recycled from cis cisternae to the ER via COPIa‐type vesicles. (vi) In S. dubia the C2 cisternae are capable of mediating the self‐assembly of scale protein complexes. (vii) In plants, ∼90% of native α‐mannosidase I localizes to medial Golgi cisternae. (viii) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre‐cis Golgi cisterna layer in mammalian cells.

Overexpression of Arabidopsis Plasmodesmata Germin-Like Proteins Disrupts Root Growth and Development

Plant cells communicate by movement of signaling agents through cytoplasmic bridges, termed plasmodesmata (PD). In this study, we characterize two members of the Germin-like protein family that are located within PD. PDGLP1/2 overexpression phenotypes had a reduction in primary root meristem size, likely due to their inability to form functional PD complexes. In plants, a population of non-cell-autonomous proteins (NCAPs), including numerous transcription factors, move cell to cell through plasmodesmata (PD). In many cases, the intercellular trafficking of these NCAPs is regulated by their interaction with specific PD components. To gain further insight into the functions of this NCAP pathway, coimmunoprecipitation experiments were performed on a tobacco (Nicotiana tabacum) plasmodesmal-enriched cell wall protein preparation using as bait the NCAP, pumpkin (Cucurbita maxima) PHLOEM PROTEIN16 (Cm-PP16). A Cm-PP16 interaction partner, Nt-PLASMODESMAL GERMIN-LIKE PROTEIN1 (Nt-PDGLP1) was identified and shown to be a PD-located component. Arabidopsis thaliana putative orthologs, PDGLP1 and PDGLP2, were identified; expression studies indicated that, postgermination, these proteins were preferentially expressed in the root system. The PDGLP1 signal peptide was shown to function in localization to the PD by a novel mechanism involving the endoplasmic reticulum-Golgi secretory pathway. Overexpression of various tagged versions altered root meristem function, leading to reduced primary root but enhanced lateral root growth. This effect on root growth was corrected with an inability of these chimeric proteins to form stable PD-localized complexes. PDGLP1 and PDGLP2 appear to be involved in regulating primary root growth by controlling phloem-mediated allocation of resources between the primary and lateral root meristems.