FG repeats facilitate integral protein trafficking to the inner nuclear membrane

The mechanism for nucleo-cytoplasmic transport of integral membrane proteins is poorly understood compared to transport of soluble molecules. We recently demonstrated that at least four distinct mechanisms can contribute to transport of integral proteins through the peripheral channels of the nuclear pore complex. One of these requires having multiple phenylalanine-glycine (FG) pairings on the integral protein. It also requires the nuclear pore complex protein Nup35, which separately contains FG repeats. FG-repeats on nuclear pore complex proteins in the central channel have been proposed to interact with FGs on transport receptors to facilitate transport of soluble proteins. Here we show that FG repeats occur quite frequently in both transmembrane and soluble proteins identified in multiple separate proteomic analyses of nuclear envelopes. We postulate that the FG repeats enable these proteins to function as their own transport receptors

The application of DamID to identify peripheral gene sequences in differentiated and primary cells

The nuclear envelope interacts extensively with chromatin, though with differences in degree and specificity in different cell types. However, identifying the specific genome sequences associated with individual nuclear envelope associated proteins, particularly nuclear membrane proteins and lamins, has been particularly difficult due to their inherent insolubility and interconnectivity. DamID is a powerful tool developed to bypass many of the inherent difficulties with identifying nuclear envelope protein-chromatin interactions and, as more tissue culture cell types derived from different tissues are examined by DamID, it is increasingly apparent that there are distinct patterns of genome organization in differentiated cell types. However, in applying DamID to both more diverse and/or differentiated cell types a number of technical caveats to the method have been observed which must be circumvented to ensure high quality data is generated. Here we elaborate a detailed methodology to adapt DamID to novel cell types, in particular differentiated cells in culture. Moreover, we highlight heretofore largely ignored variations in the PCR amplified DNA products generated by the DamID procedure and the consequences they have for downstream analysis steps. Thus, the methods described here should serve as a useful resource to researchers new to DamID as well as readily allow its application to an expanded set of cell types and conditions

NETs and cell cycle regulation

There are many ways that the nuclear envelope can influence the cell cycle. In addition to roles of lamins in regulating the master cell cycle regulator pRb and nuclear envelope breakdown in mitosis, many other nuclear envelope proteins influence the cell cycle through regulatory or structural functions. Of particular note among these are the nuclear envelope transmembrane proteins (NETs) that appear to influence cell cycle regulation through multiple separate mechanisms. Some NETs and other nuclear envelope proteins accumulate on the mitotic spindle, suggesting functional or structural roles in the cell cycle. In interphase exogenous overexpression of some NETs promotes an increase in G1 populations, while others promote an increase in G2/M populations, sometimes associated with the induction of senescence. Intriguingly, most of the NETs linked to the cell cycle are highly restricted in their tissue expression; thus, their misregulation in cancer could contribute to the many tissue-specific types of cancer

Migraine-associated common genetic variants confer greater risk of posterior vs. anterior circulation ischemic stroke

To examine potential genetic relationships between migraine and the two distinct phenotypes posterior circulation ischemic stroke (PCiS) and anterior circulation ischemic stroke (ACiS), we generated migraine polygenic risk scores (PRSs) and compared these between PCiS and ACiS, and separately vs. non-stroke control subjects. Acute ischemic stroke cases were classified as PCiS or ACiS based on lesion location on diffusion-weighted MRI. Exclusion criteria were lesions in both vascular territories or uncertain territory; supratentorial PCiS with ipsilateral fetal posterior cerebral artery; and cases with atrial fibrillation. We generated migraine PRS for three migraine phenotypes (any migraine; migraine without aura; migraine with aura) using publicly available GWAS data and compared mean PRSs separately for PCiS and ACiS vs. non-stroke control subjects, and between each stroke phenotype. Our primary analyses included 464 PCiS and 1079 ACiS patients with genetic European ancestry. Compared to non-stroke control subjects (n=15396), PRSs of any migraine were associated with increased risk of PCiS (p=0.01–0.03) and decreased risk of ACiS (p=0.010–0.039). Migraine without aura PRSs were significantly associated with PCiS (p=0.008–0.028), but not with ACiS. When comparing PCiS vs. ACiS directly, migraine PRSs were higher in PCiS vs. ACiS for any migraine (p=0.001–0.010) and migraine without aura (p=0.032–0.048). Migraine with aura PRS did not show a differential association in our analyses. Our results suggest a stronger genetic overlap between unspecified migraine and migraine without aura with PCiS compared to ACiS. Possible shared mechanisms include dysregulation of cerebral vessel endothelial function