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.     

Interplay between microtubule dynamics and intracellular organization

Microtubules are hollow tubes essential for many cellular functions such as cell polarization and migration, intracellular trafficking and cell division. They are polarized polymers composed of α and β tubulin that are, in most cells, nucleated at the centrosome at the center of the cell. Microtubule plus-ends are oriented towards the periphery of the cell and explore the cytoplasm in a very dynamic manner. Microtubule alternate between phases of growth and shrinkage in a manner described as dynamic instability. Their dynamics is highly regulated by multiple factors: tubulin post-translational modifications such as detyrosination or acetylation, and microtubule-associated proteins, among them the plus-tip tracking proteins. This regulation is necessary for microtubule functions in the cell. In this review, we will focus on the role of microtubules in intracellular organization. After an overview of the mechanisms responsible for the regulation of microtubule dynamics, the major roles of microtubules dynamics in organelle positioning and organization in interphase cells will be discussed. Conversely, the role of certain organelles, like the nucleus and the Golgi apparatus as microtubule organizing centers will be reviewed. We will then consider the role of microtubules in the establishment and maintenance of cell polarity using few examples of cell polarization: epithelial cells, neurons and migrating cells. In these cells, the microtubule network is reorganized and undergoes specific and local regulation events; microtubules also participate in the intracellular reorganization of different organelles to ensure proper cell differentiation.     

Mitochondria drive autophagy pathology via microtubule disassembly

Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle “quality” allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients’ mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD.     

Re-evaluation of physical interaction between plant peroxisomes and other organelles using live-cell imaging techniques

The dynamic behavior of organelles is essential for plant survival under various environmental conditions. Plant organelles, with various functions,migrate along actin filaments and contact other types of organelles, leading to physical interactions at a specific site called the membrane contact site. Recent studies have revealed the importance of physical interactions in maintaining efficient metabolite flow between organelles.In this review, we first summarize peroxisome function under different environmental conditions and growth stages to understand organelle interactions. We then discuss current knowledge regarding the interactions between peroxisome and other organelles, i.e., the oil bodies, chloroplast, and mitochondria from the perspective of metabolic and physiological regulation, with reference to various organelle interactions and techniques for estimating organelle interactions occurring in plant cells.     

Intermediate Filaments and Vesicular Membrane Traffic: The Odd Couple's First Dance?

During the last two decades, much attention has been focused on the regulation of membrane traffic by the actin and microtubule cytoskeletal networks. Their dynamic and polarized behavior and associated motors provide a logical framework from which architectural and movement cues can be communicated to organelles. The study of these cytoskeletal systems has been greatly aided by pharmacological agents. In contrast, intermediate filaments (IFs) have largely been neglected as a potential player in membrane traffic, both because a comprehensive pharmacology to perturb them does not exist and because they lack the intrinsic polarity and specific motors that make the other cytoskeletal systems attractive. In this review, we will discuss evidence suggesting that IFs may play roles in controlling organelle positioning and in membrane protein targeting. Furthermore, we will discuss potential mechanisms by which IFs may regulate the localization and function of organelles.     

Atg5 regulates late endosome and lysosome biogenesis

Autophagy is an evolutionarily conserved lysosome-based degradation process.Atg5 plays a very important role in autophagosome formation.Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner.In Atg5 cells,but not in other essential autophagy genes defecting cells,recycling and retrieval of late endosomal components from hybrid organelles are impaired,causing persistent hybrid organelles and defective formation of late endosomes and lysosomes.Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles.Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 cells.Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.     

Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P-binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod-specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10-deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.     

Organelle distribution in neurons: Logistics behind polarized transport

Highly polarized neurons need to carefully regulate the distribution of organelles and other cargoes into their two morphologically and functionally distinct domains, the somatodendritic and axonal compartments, to maintain proper neuron homeostasis. An outstanding question in the field is how organelles reach their correct destination. Long-range transport along microtubules, driven by motors, ensures a fast and controlled availability of organelles in axons and dendrites, but it remains largely unclear what rules govern their transport into the correct compartment. Here, we review the emerging concepts of polarized cargo trafficking in neurons, highlighting the role of microtubule organization, microtubule-associated proteins, and motor proteins and discuss compartment-specific inclusion and exclusion mechanisms as well as the regulation of correct coupling of cargoes to motor proteins.     

The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis

The dynamic equilibrium between vesicle fission and fusion at Golgi, endosome, and vacuole/lysosome is critical for the maintenance of organelle identity. It depends, among others, on Rab GTPases and tethering factors, whose function and regulation are still unclear. We now show that transport among Golgi, endosome, and vacuole is controlled by two homologous tethering complexes, the previously identified HOPS complex at the vacuole and a novel endosomal tethering (CORVET) complex, which interacts with the Rab GTPase Vps21. Both complexes share the four class C Vps proteins: Vps11, Vps16, Vps18, and Vps33. The HOPS complex, in addition, contains Vps41/Vam2 and Vam6, whereas the CORVET complex has the Vps41 homolog Vps8 and the (h)Vam6 homolog Vps3. Strikingly, the CORVET and HOPS complexes can interconvert; we identify two additional intermediate complexes, both consisting of the class C core bound to Vam6-Vps8 or Vps3-Vps41. Our data suggest that modular assembled tethering complexes define organelle biogenesis in the endocytic pathway.     

Maintaining social contacts: The physiological relevance of organelle interactions

Membrane-bound organelles in eukaryotic cells form an interactive network to coordinate and facilitate cellular functions. The formation of close contacts, termed “membrane contact sites” (MCSs), represents an intriguing strategy for organelle interaction and coordinated interplay. Emerging research is rapidly revealing new details of MCSs. They represent ubiquitous and diverse structures, which are important for many aspects of cell physiology and homeostasis. Here, we provide a comprehensive overview of the physiological relevance of organelle contacts. We focus on mitochondria, peroxisomes, the Golgi complex and the plasma membrane, and discuss the most recent findings on their interactions with other subcellular organelles and their multiple functions, including membrane contacts with the ER, lipid droplets and the endosomal/lysosomal compartment.     

Kinesin-2 is a motor for late endosomes and lysosomes

The bidirectional nature of late endosome/lysosome movement suggests involvement of at least two distinct motors, one minus-end directed and one plus-end directed. Previous work has identified dynein as the minus-end-directed motor for late endosome/lysosome localization and dynamics. Conventional kinesin (kinesin-1) has been implicated in plus-end-directed late endosome/lysosome movement, but other kinesin family members may also be involved. Kinesin-2 is known to drive the movement of pigment granules, a type of lysosomally derived organelle, and was recently found to be associated with purified late endosomes. To determine whether kinesin-2 might also power endosome movement in non-pigmented cells, we overexpressed dominant negative forms of the KIF3A motor subunit and KAP3 accessory subunit and knocked down KAP3 levels using RNAi. We found kinesin-2 to be required for the normal steady-state localization of late endosomes/lysosomes but not early endosomes or recycling endosomes. Despite the abnormal subcellular distribution of late endosomes/lysosomes, the uptake and trafficking of molecules through the conventional endocytic pathway appeared to be unaffected. The slow time-course of inhibition suggests that both kinesin-2 itself and its attachment to membranes do not turn over quickly.     

Method of modelling intracellular transport in branching neurites: application to axons and dendrites of Drosophila sensory neurons

This paper develops a method of calculating the transport of intracellular organelles in neurons with branching neurites which is based on the Smith–Simmons equations of motor-assisted transport. The method is aimed at understanding the effects of microtubule (MT) polarity orientation in branching neurites on transport of organelles at the fundamental level. The method is applied to calculating the organelle transport in axons and dendrites of Drosophila neurons, using the map of MT orientation in such neurons developed by Stone et al. (Mol Biol Cell 19:4122–4129, 2008). The proximal dendrite is assumed to branch and form two distal dendrites. Two different MT polarity arrangements in a proximal dendrite are considered, and implications of these MT arrangements on organelle transport are analysed. It is demonstrated that the MT arrangement found in Drosophila dendrites (MTs have their minus ends out in a proximal dendrite) results in much more efficient motor-driven transport than the structure with a mixed MT orientation in proximal dendrites.     

Lysosome Transport as a Function of Lysosome Diameter

Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter.     

Rather rule than exception? How to evaluate the relevance of dual protein targeting to mitochondria and chloroplasts

Dual targeting of a nuclearly encoded protein into two different cell organelles is an exceptional event in eukaryotic cells. Yet, the frequency of such dual targeting is remarkably high in case of mitochondria and chloroplasts, the two endosymbiotic organelles of plant cells. In most instances, it is mediated by “ambiguous” transit peptides, which recognize both organelles as the target. A number of different approaches including in silico, in organello as well as both transient and stable in vivo assays are established to determine the targeting specificity of such transit peptides. In this review, we will describe and compare these approaches and discuss the potential role of this unusual targeting process. Furthermore, we will present a hypothetical scenario how dual targeting might have arisen during evolution.     

Dual targeting to mitochondria and chloroplasts.

Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.     

Why just eat in,when you can also eat out?

The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).