Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-β that is distinct from that used to bind importin-α

N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

© 2018 National Academy of Sciences. All Rights Reserved. The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington’s disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA ad-ducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems

Practical three color live cell imaging by widefield microscopy

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope

Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin–actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin–actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement–Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin–cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases

Future ready: Strengthening oncology nursing leadership in the context of professional oncology nursing organizations [Guest Editorial]

Every nurse is a leader. Rather than being situated only within a traditional leadership role or title, nursing leadership is about critical thinking, action and advocacy across all roles, practice settings and domains of nursing practice. When high-quality nursing leadership is enacted, positive patient, provider and system outcomes are demonstrated (Cummings et al., 2010; National Expert Commission, 2012; Wong, Cummings & Ducharme, 2013). Leadership begins in undergraduate education and continues throughout one’s career. Nursing leadership in this context is about looking beyond nursing, as a series of scientific acts of caring that can change individual lives, to include lifelong commitment to political action for system change. It requires nurses to lift their gaze from focusing only on individuals to populations, and from the local to global context (Canadian Nurses Association, 2009; Institute of Medicine, 2011; International Council of Nurses, 2016; National Expert Commission, 2012)

ROS-specific Huntingtin Interactions: GFP reactivation assay optimization in HEK 293 cells

Optimization step in the lead up to phenotypic analysis of ROS-dependent huntingtin interacting proteins

ROS-specific Huntingtin Interactions: Fractionation Optimization

Optimization step in the lead up to mass spec identification of ROS-specific huntingtin protein-protein interactions

ROS-specific Huntingtin Interactions: GFP reactivation assay WT vs HD mouse striatal cells

Optimization step in the lead up to phenotypic analysis of ROS-dependent huntingtin interacting proteins