Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles

Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells.     

Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.     

Loss of Dja2 accompanies pH deviation in lysosomes and lysosome-related organelles

The Dja2 knockout (Dja2^−/−) mice had respiratory distress, and >60% died within 2 days after birth. The surviving adult Dja2^−/− mice were infertile and the lungs of Dja2^−/− mice showed several abnormalities, including the processing defect of prosurfactant protein C in the alveolar epithelial type II cells and the accumulation of glycolipids in enlarged alveolar macrophages. The luminal pH of acidic organelles in Dja2^−/− cells was shifted to pH 5.37–5.45. This deviated pH was immediately restored to control levels (pH 4.56–4.65) by the addition of a diuretic, ethyl isopropyl amiloride (EIPA). Although the role of DJA2 in maintaining the pH homeostasis of lysosome-related organelles is currently obscure, this rapid and remarkable pH resilience is best explained by an EIPA-sensitive proton efflux machinery that is disorganized and overactivated due to the loss of Dja2.     

Interaction of Hermansky-Pudlak Syndrome genes in the regulation of lysosome-related organelles

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease caused by abnormalities in the synthesis and/or trafficking of lysosome-related organelles (LROs) including melanosomes, lamellar bodies of lung type II cells and platelet dense granules. At least 15 genes cause HPS in mice, with a significant number specifying novel subunits of protein complexes termed BLOCs (Biogenesis of Lysosome-related Organelles Complexes). To ascertain whether BLOC complexes functionally interact in vivo, mutant mice doubly or triply deficient in protein subunits of the various BLOC complexes and/or the AP-3 adaptor complex were constructed and tested for viability and for abnormalities of melanosomes, lung lamellar bodies and lysosomes. All mutants, including those deficient in all three BLOC complexes, were viable though the breeding efficiencies of multiple mutants involving AP-3 were severely compromised. Interactions of BLOC protein complexes with each other and with AP-3 to affect most LROs were apparent. However, these interactions were tissue and organelle dependent. These studies document novel biological interactions of BLOC and AP-3 complexes in the biosynthesis of LROs and assess the role(s) of HPS protein complexes in general health and physiology in mammals. Double and triple mutant HPS mice provide unique and practical experimental advantages in the study of LROs.     

Pallidin is a component of a multi-protein complex involved in the biogenesis of lysosome-related organelles

The Hermansky–Pudlak syndrome defines a group of genetic disorders characterized by defective lysosome-related organelles such as melanosomes and platelet dense bodies. Hermansky–Pudlak syndrome can be caused by mutations of at least four genes in humans and 15 genes in mice. One of these genes is mutated in the pallid mouse strain and encodes a novel protein named pallidin (L. Huang, Y. M. Kuo and J. Gitschier, Nat Genet 1999; 23: 329–332). Pallidin has no homology to any other known protein and no recognizable functional motifs. We have conducted a biochemical characterization of human pallidin using a newly developed polyclonal antibody. We show that pallidin is a ubiquitously expressed ∼ 25 kDa protein found both in the cytosol and peripherally associated to membranes. Sedimentation velocity analyses show that native pallidin has a sedimentation coefficient of ∼ 5.1 S, much larger than expected from the molecular mass of the pallidin polypeptide. In line with this observation, cosedimentation and coprecipitation analyses reveal that pallidin is part of a hetero-oligomeric complex. One of the subunits of this complex is the product of another Hermansky–Pudlak syndrome gene, muted. Fibroblasts derived from the muted mouse strain exhibit reduced levels of pallidin, suggesting that the absence of the muted protein destabilizes pallidin. These observations indicate that pallidin is a subunit of a novel multi-protein complex involved in the biogenesis of lysosome-related organelles.     

Biogenesis of lamellar bodies,lysosome-related organelles involved in storage and secretion of pulmonary surfactant

Lamellar bodies are members of a subclass of lysosome-related organelles referred to as secretory lysosomes. The principal constituents of the lamellar body, surfactant phospholipids, are organized into tightly packed, bilayer membranes in a process that is strongly influenced by the lung-specific, hydrophobic peptide SP-B. Newly synthesized SP-B is transported from the Golgi to the lamellar body via multivesicular bodies; in contrast, preliminary evidence suggests that newly synthesized surfactant phospholipids are transported from the ER and incorporated into the internal membranes of the lamellar body via a distinct pathway.     

Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.     

Dendritic spine morphogenesis and plasticity

Dendritic spines are small protrusions off the dendrite that receive excitatory synaptic input. Spines vary in size, likely correlating with the strength of the synapses they form. In the developing brain, spines show highly dynamic behavior thought to facilitate the formation of new synaptic contacts. Recent studies have illuminated the numerous molecules regulating spine development, many of which converge on the regulation of actin filaments. In addition, interactions with glial cells are emerging as important regulators of spine morphology. In many cases, spine morphogenesis, plasticity, and maintenance also depend on synaptic activity, as shown by recent studies demonstrating changes in spine dynamics and maintenance with altered sensory experience.     

Primary cilia are WNT-transducing organelles whose biogenesis is controlled by a WNT-PP1 axis

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The BLOS1-interacting protein KXD1 is involved in the biogenesis of lysosome-related organelles

Biogenesis of lysosome‐related organelles (LROs) complex‐1 (BLOC‐1) is an eight‐subunit complex involved in lysosomal trafficking. Interacting proteins of these subunits expand the understanding of its biological functions. With the implementation of the naïve Bayesian analysis, we found that a human uncharacterized 20 kDa coiled‐coil KxDL protein, KXD1, is a BLOS1‐interacting protein. In vitro binding assays confirmed the interaction between BLOS1 and KXD1. The mouse KXD1 homolog was widely expressed and absent in Kxd1 knockout (KO) mice. BLOS1 was apparently reduced in Kxd1‐KO mice. Mild defects in the melanosomes of the retinal pigment epithelia and in the platelet dense granules of the Kxd1‐KO mouse were observed, mimicking a mouse model of mild Hermansky–Pudlak syndrome that affects the biogenesis of LROs.     

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.     

Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins

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Diffusion resistances between ADH-induced vacuoles and the extracellular space in rabbit collecting duct: evidence that mos vacuoles are intracellular,endocytic compartments

Summary Large vacuoles form in the renal collecting duct following the onset of antidiuretic hormone (ADH)-stimulated water reabsorption. The aim of the present study was to test two alternative hypotheses regarding the origins of these structures: (1) the vacuoles constitute basilar, extracellular spaces that dilate as water flows through these spaces from cells into the peritubular compartment; or (2) the vacuoles represent intracellular, endocytic compartments that dilate during water reabsorption due to enhanced fluid phase endocytosis. Fluorescence-digital imaging microscopy was used to visualize the uptake into vacuoles of a hydrophilic fluorochrome (6 methoxy-N-[3 sulfopropyl] quinolinium) whose fluorescence is markedly quenched by halides. During their formation, most vacuoles (67%) accumulated the fluorochrome from the peritubular bath and trapped the dye well after (>60 min) washing it from the bath. The spatial pattern of fluorescence within individual vacuoles indicated that the dye was trapped within these structures as a fluid-phase marker and was not bound to the vacuole margins. The fluorescence of dye trapped within vacuoles was virtually unaltered by changes in peritubular Cl^- or Br^- concentration that elicit dramatic quenching of dye-fluorescence in bulk solution, as expected if there exists a high diffusion resistance between the interiors of these structures and the peritubular space. These results indicate that most ADH-induced vaculles represent endocytic compartments that are not directly connected to the extracellular space.     

Hair follicle stem cells are specified and function in early skin morphogenesis

In adult skin, epithelial hair follicle stem cells (SCs) reside in a quiescent niche and are essential for cyclic bouts of hair growth. Niche architecture becomes pronounced postnatally at the start of the first hair cycle. Whether SCs exist or function earlier is unknown. Here we show that slow-cycling cells appear early in skin development, express SC markers, and later give rise to the adult SC population. To test whether these early slow-cycling cells function as SCs, we use Sox9-Cre for genetic marking and K14-Cre to embryonically ablate Sox9, an essential adult SC gene. We find that the progeny of Sox9-expressing cells contribute to all skin epithelial lineages and Sox9 is required for SC specification. In the absence of early SCs, hair follicle and sebaceous gland morphogenesis is blocked, and epidermal wound repair is compromised. These findings establish the existence of early hair follicle SCs and reveal their physiological importance in tissue morphogenesis.     

Lysosome and lysosome-related organelles responsible for specialized functions in higher organisms, with special emphasis on vacuolar-type proton ATPase

Mammals contain various cells differentiated in both morphology and function, which play vital roles in tissue-specific functions. Late endosome/lysosome and lysosomal-related organelles are involved in these specialized functions including antigen presentation, bone remodeling and hormone regulation. To fulfill these diverse roles, lysosomes are present at different levels in different tissues and cell types; however, their morphology within these different tissues varies and the regulation of their activities differs with lysosomal compartments in some cells also functioning as secretory compartments. The luminal acidification of these organelles is closely correlated with their functions. This review will discuss the functions of lysosomes and lysosomal-related organelles, with particular emphasis on the major proton pump, the vacuolar-type proton ATPase (V-ATPase), which is responsible for luminal acidification.     

NAADP mobilizes Ca(2+) from reserve granules,lysosome-related organelles,in sea urchin eggs

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca(2+) in many cells and species. Unlike other Ca(2+)-mobilizing messengers, NAADP mobilizes Ca(2+) from an unknown store that is not the endoplasmic reticulum, the store traditionally associated with messenger-mediated Ca(2+) signaling. Here, we demonstrate the presence of a Ca(2+) store in sea urchin eggs mobilized by NAADP that is dependent on a proton gradient maintained by an ATP-dependent vacuolar-type proton pump. Moreover, we provide pharmacological and biochemical evidence that this Ca(2+) store is the reserve granule, the functional equivalent of a lysosome in the sea urchin egg. These findings represent an unsuspected mechanism for messenger-mediated Ca(2+) release from lysosome-related organelles.     

Evidence that the neck appendages are adsorption organelles in Bacillus subtilis bacteriophage phi29.

A mutant of Bacillus subtilis unable to adsorb phage phi29 efficiently has been isolated. This mutant can be infected by host range mutants of the phage. Since the host range mutations map in cistron 12, which codes for neck appendage protein, this would tend to confirm that these organelles are involved in viral adsorption.     

Caenorhabditis elegans HOPS and CCZ-1 mediate trafficking to lysosome-related organelles independently of RAB-7 and SAND-1

As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome–lysosome fusion and the consumption of AP-3–containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type–specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1–related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences.