Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling.

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15-20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins.

Hereditary spastic paraplegia (HSP) is a neurological syndrome characterized by degeneration of central nervous system (CNS) axons. Mutated HSP proteins include myelin proteolipid protein (PLP) and axon-enriched proteins involved in mitochondrial function, smooth endoplasmic reticulum (SER) structure, and microtubule (MT) stability/function. We characterized axonal mitochondria, SER, and MTs in rodent optic nerves where PLP is replaced by the peripheral nerve myelin protein, P0 (P0-CNS mice). Mitochondrial pathology and degeneration were prominent in juxtaparanodal axoplasm at 1 mo of age. In wild-type (WT) optic nerve axons, 25% of mitochondria-SER associations occurred on extensions of the mitochondrial outer membrane. Mitochondria-SER associations were reduced by 86% in 1-mo-old P0-CNS juxtaparanodal axoplasm. 1-mo-old P0-CNS optic nerves were more sensitive to oxygen-glucose deprivation and contained less adenosine triphosphate (ATP) than WT nerves. MT pathology and paranodal axonal ovoids were prominent at 6 mo. These data support juxtaparanodal mitochondrial degeneration, reduced mitochondria-SER associations, and reduced ATP production as causes of axonal ovoid formation and axonal degeneration.

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles.

Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.