Mitochondrial dysfunction has been associated with schizophrenia (SZ) and bipolar disorder (BD). This review examines recent publications and novel associations between mitochondrial genes and SZ and BD. Associations of nuclear-encoded mitochondrial variants with SZ were found using gene- and pathway-based approaches. Two control region mitochondrial DNA (mtDNA) SNPs, T16519C and T195C, both showed an association with SZ and BD. A review of 4 studies of A15218G located in the cytochrome B oxidase gene (CYTB, SZ = 11,311, control = 35,735) shows a moderate association with SZ ( = 2.15E-03). Another mtDNA allele A12308G was nominally associated with psychosis in BD type I subjects and SZ. The first published study testing the epistatic interaction between nuclear-encoded and mitochondria-encoded genes demonstrated evidence for potential interactions between mtDNA and the nuclear genome for BD. A similar analysis for the risk of SZ revealed significant joint effects (34 nuclear-mitochondria SNP pairs with joint effect ≤ 5E-07) and significant enrichment of projection neurons. The mitochondria-encoded gene CYTB was found in both the epistatic interactions for SZ and BD and the single SNP association of SZ. Future efforts considering population stratification and polygenic risk scores will test the role of mitochondrial variants in psychiatric disorders.

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

Metazoans have evolved multiple paralogues of the TATA binding protein (TBP), adding another tunable level of gene control at core promoters. While TBP-related factor 1 (TRF1) shares extensive homology with TBP and can direct both Pol II and Pol III transcription in vitro, TRF1 target sites in vivo have remained elusive. Here, we report the genome-wide identification of TRF1-binding sites using high-resolution genome tiling microarrays. We found 354 TRF1-binding sites genome-wide with approximately 78% of these sites displaying colocalization with BRF. Strikingly, the majority of TRF1 target genes are Pol III-dependent small noncoding RNAs such as tRNAs and small nonmessenger RNAs. We provide direct evidence that the TRF1/BRF complex is functionally required for the activity of two novel TRF1 targets (7SL RNA and small nucleolar RNAs). Our studies suggest that unlike most other eukaryotic organisms that rely on TBP for Pol III transcription, in Drosophila and possibly other insects the alternative TRF1/BRF complex appears responsible for the initiation of all known classes of Pol III transcription.

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant.

The principal regulator of p53 stability is HDM2, an E3 ligase that mediates p53 degradation via the ubiquitin-26S proteasome pathway. The current model holds that p53 degradation occurs exclusively on cytoplasmic proteasomes and hence has an absolute requirement for nuclear export of p53 via the CRM-1 pathway. However, proteasomes are abundant in both cytosol and nucleus, and no studies have been done to determine under what physiological circumstances p53 degradation might occur in the nucleus. We analyzed HDM2-mediated degradation of endogenous p53 in the presence of various nuclear export inhibitors of CRM-1, including leptomycin B (LMB), a noncompetitive, specific, and fast-acting inhibitor; and HTLV1-Rex protein, a potent competitive inhibitor. We found that significant HDM2-mediated p53 degradation took place in the presence of LMB or HTLV1-Rex, indicating that endogenous p53 degradation occurs locally in the nucleus, in parallel to cytoplasmic degradation. Moreover, p53 null cells that coexpressed export-defective mutants of p53 and HDM2 retained partial competence for p53 degradation. It is important that nuclear degradation of p53 occurred during the poststress recovery phase of a p53 response, after DNA damage ceased. We propose that the capability of local p53 degradation within the nucleus provides a tighter and faster control during the down-regulatory phase, when an active p53 program needs to be turned off quickly.

SignificanceInteractions between the cell nucleus and cytoskeleton regulate cell mechanics and are facilitated by the interplay between the nuclear lamina and linker of nucleoskeleton and cytoskeleton (LINC) complexes. To date, the specific contribution of the four lamin isoforms to nucleocytoskeletal connectivity and whole-cell mechanics remains unknown. We discover that A- and B-type lamins distinctively interact with LINC complexes that bind F-actin and vimentin filaments to differentially modulate cortical stiffness, cytoplasmic stiffness, and contractility of mouse embryonic fibroblasts (MEFs). We propose and experimentally verify an integrated lamin-LINC complex-cytoskeleton model that explains cellular mechanical phenotypes in lamin-deficient MEFs. Our findings uncover potential mechanisms for cellular defects in human laminopathies and many cancers associated with mutations or modifications in lamin isoforms.

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

The internal workings of the nucleus remain a mystery. A list of component parts exists, and in many cases their functional roles are known for events such as transcription, RNA processing, or nuclear export. Some of these components exhibit structural features in the nucleus, regions of concentration or bodies that have given rise to the concept of functional compartmentalization–that there are underlying organizational principles to be described. In contrast, a picture is emerging in which transcription appears to drive the assembly of the functional components required for gene expression, drawing from pools of excess factors. Unifying this seemingly dual nature requires a more rigorous approach, one in which components are tracked in time and space and correlated with onset of specific nuclear functions. In this chapter, we anticipate tools that will address these questions and provide the missing kinetics of nuclear function. These tools are based on analyzing the fluctuations inherent in the weak signals of endogenous nuclear processes and determining values for them. In this way, it will be possible eventually to provide a computational model describing the functional relationships of essential components.

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3σ) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCCs) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins.