Practical Considerations for Using Pooled Lentiviral CRISPR Libraries.

CRISPR/Cas9 technology is ideally suited for genome-wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available, but while using CRISPR/Cas9 for genetic experiments has become widely adopted, genome-wide screening experiments remain technically challenging. This review covers the basics of CRISPR/Cas9, describes several publicly available CRISPR libraries, and provides a general protocol for conducting genome-wide screening experiments using CRISPR/Cas9. © 2016 by John Wiley & Sons, Inc.

Αvß3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway.

Protein transport through the endosome is critical for maintaining proper integrin cell surface integrin distribution to support cell adhesion, motility and viability. Here we employ a live-cell imaging approach to evaluate the relationship between integrin function and transport through the early endosome. We discovered that two early endosome factors, AAK1L and EHD3, are critical for αvß3-integrin-mediated cell adhesion in HeLa cells. siRNA-mediated depletion of either factor delays short-loop ß3 integrin recycling from the early endosome back to the cell surface. Total internal reflection fluorescence-based colocalization analysis reveals that ß3 integrin transits AAK1L- and EHD3-positive endosomes near the cell surface, a subcellular location consistent with a rapid-recycling role for both factors. Moreover, structure-function analysis reveals that AAK1L kinase activity, as well as its C-terminal domain, is essential for cell adhesion maintenance. Taken together, these data reveal an important role for AAK1L and EHD3 in maintaining cell viability and adhesion by promoting αvß3 integrin rapid recycling from the early endosome.

Notch signaling from the endosome requires a conserved dileucine motif.

Notch signaling is reliant on γ-secretase-mediated processing, although the subcellular location where γ-secretase cleaves Notch to initiate signaling remains unresolved. Accumulating evidence demonstrates that Notch signaling is modulated by endocytosis and endosomal transport. In this study, we investigated the relationship between Notch transport itinerary and signaling capacity. In doing so, we discovered a highly conserved dileucine sorting signal encoded within the cytoplasmic tail that directs Notch to the limiting membrane of the lysosome for signaling. Mutating the dileucine motif led to receptor accumulation in cation-dependent mannose-phosphate receptor-positive tubular early endosomes and a reduction in Notch signaling capacity. Moreover, truncated receptor forms that mimic activated Notch were readily cleaved by γ-secretase within the endosome; however, the cleavage product was proteasome-sensitive and failed to contribute to robust signaling. Collectively these results indicate that Notch signaling from the lysosome limiting membrane is conserved and that receptor targeting to this compartment is an active process. Moreover, the data support a model in which Notch signaling in mammalian systems is initiated from either the plasma membrane or lysosome, but not the early endosome.

Structure and function of epididymal protein cysteine-rich secretory protein-1.

Cysteine-rich secretory protein-1 (CRISP-1) is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP-1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract.

Glutamate-related gene expression changes with age in the mouse auditory midbrain.

Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.