Sperm secrets finally revealed

It has long been known that sperm cells undergo a remarkable change, called hyperactivation, upon entering the alkaline environment of the female reproductive tract. A sperm's tail begins to beat in a frantic whip-like motion, which can help propel it into an egg for fertilization. This is thought to be brought about by an increase in calcium in the flagellum, but researchers wanting to study the responsible ion channels have been thwarted for decades by the difficulty of patch-clamping the tight membrane that surrounds the stiff intracellular structures. Now, Clapham and colleagues have developed a reproducible technique for whole-cell recordings of sperm. They managed to patch-clamp remnant cytoplasmic droplets that are found on immature sperm but are shed upon ejaculation. These droplets are attached to the stiff underlying structures of the flagella and allow a pipette to form a tight seal with the sperm's membrane. Using this method, they showed that CatSper1, a protein known to be essential for male fertility, is a key component of a flagellar calcium channel. Armed with this technique, researchers can begin to probe how calcium leads to structural changes in the flagella, as well as many other aspects of sperm machinery, in exquisite detail. (Nature 439, 737–740, 2006). TSS

Dicer's close associates

Silencing of gene expression through RNA-mediated interference (RNAi) involves different types of small RNAs, such as short interfering RNAs (siRNAs) and microRNAs (miRNAs). These small RNAs are generated from both exogenous and endogenous dsRNA precursors and exert their silencing activities through multiple mechanisms. The processing of different classes of dsRNA precursors into siRNAs or miRNAs requires members of the Dicer family of proteins. This raises the question of how Dicer recognizes different classes of dsRNA and how the processed RNA products are incorporated into the correct RNAi pathways. To begin to answer this question, Mello and colleagues have used a proteomics approach to identify proteins that interact with Dicer (DCR-1) in Caenorhabditis elegans. Immunoprecipitation confirmed ∼20 interactors, and the phenotypes of individual deletions of 15 of these were further investigated. The results provide insight into the processes that require these interactors. For example, the homolog of an RNA phosphatase called PIR-1 is essential for the accumulation of siRNAs. In addition, the authors uncovered two new 'enhancer of RNAi' genes, eri-3 and eri-5, and showed that the products of these eri genes may promote the assembly of small RNA-processing complexes. Finally, the authors observed that some of the interactors are involved in the biogenesis of specific classes of small RNAs, but the different pathways influence one another, suggesting that these pathways may compete for Dicer or other limiting downstream silencing factors. These observations pave the way toward a molecular understanding of how specific small RNAs enter the different RNAi pathways. (Cell 124, 343–354, 2006) DM

The Derlins of degradation

The endoplasmic reticulum (ER) is the staging area for secreted proteins. It contains a quality-control system to ensure that only correctly folded proteins are secreted. When a large number of unfolded or misfolded proteins are present in the ER, it induces an unfolded protein response (UPR) to enhance the cell's capacity to handle such stress. The response increases the production of ER-resident molecular chaperones and the components of the ER-associated degradation (ERAD) pathway, which destroys polypeptides that fail to fold even with the help of molecular chaperones. The key components in the ERAD pathway include an ER protein called EDEM, which recognizes misfolded glycoproteins destined for degradation, and a cytosolic ATPase called p97, which extracts these proteins from the ER. The molecular links between EDEM and p97 have been clearly defined. Mori and colleagues now show that two mammalian ER-membrane proteins, Derlin-2 and Derlin-3, connect the ERAD components in the ER and the cytosol. Derlin-2 and Derlin-3 are homologs of Derlin-1, a protein involved in degrading the major histocompatibility complex class I heavy chain induced by human cytomegalovirus in a mechanism similar to that of ERAD. The authors show that the expression of Derlin-2 and Derlin-3 is regulated by UPR; in particular, the same regulatory pathway of UPR controls the expression of Derlin-2 and EDEM. Both Derlin-2 and Derlin-3 are localized in the ER, associate with p97 and EDEM, and are required for the degradation of misfolded glycoproteins via ERAD. It remains to be determined whether the Derlins form a protein-conducting channel for substrate retrotranslocation. (J. Cell Biol. 172, 383–393, 2006). HPF

Dealing with defects

DNA damage comes in different shapes and sizes, so most cells have different repair systems to deal with the damage. One pathway, known as nucleotide excision repair (NER), can remove a number of structurally unrelated lesions, including UV-induced damage. Defects in NER lead to the UV-sensitive cancer-prone disorder xeroderma pigmentosum (XP). Seven XP complementation groups (XP-A through G) in the NER pathway have been genetically defined. Cells in the XP-E group have a defect in the recognition of UV-damaged DNA. Biochemically, mutations causing this defect are mapped onto a protein complex called UV-DDB. UV-DDB has two subunits, DDB1 and DDB2, and is a component of the newly identified cullin 4A-based ubiquitin (Ub) E3 ligase, DDB1-CUL4ADDB2 E3 ligases regulate protein activity or target proteins for degradation by the proteasomal pathway. To understand more about how DDB1-CUL4ADDB2 E3 brings about DNA repair, Rapi´c-Otrin and colleagues looked for an in vivo target of this ligase. They found that DDB1-CUL4ADDB2 E3 colocalizes with UV-damaged DNA, but in XP-E cells, the complex fails to assemble and does not bind chromatin after UV irradiation. They further showed that DDB1-CUL4ADDB2 E3 targets histone H2A for monoubiquitination at sites of UV damage. These findings suggest that the ubiquitination of H2A could make the chromatin more accessible to the repair machinery or could act as a signal for the recruitment of NER factors. Further experiments to specifically pin down the role of the Ub-H2A modification should help answer the general question of how the repair machinery recognizes and gains access to DNA damage within the context of chromatin. (Proc. Natl. Acad. Sci. USA 103, 2588–2593, 2006). BK

Research highlights written by Hwa-ping Feng, Boyana Konforti, Dorothy Moore and Tracy Smith Schmidt.