In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

The mechanisms driving pathological beta-amyloid (Ab) generation in late-onset Alzheimer's disease (AD) are unclear. Two late-onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Ab generation in neurons. We identify a novel neuron-specific polarisation of Ab generation controlled by Bin1 and CD2AP. We discover that Bin1 and CD2AP control Ab generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Ab generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Ab in neurons increasing the risk for late-onset AD.

Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca2+, H+, K+, and Cl− -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H+-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H+ effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H+ flux profiles. Furthermore, there is no net flux of H+ in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes.

SUMMARYThe importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffer´s vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffer´s vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP‐tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse‐chase and quench‐chase‐pulse experiments. These time‐slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP‐tagging in fixed‐ and live‐cell studies and evaluate the recently developed fast‐acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification. Curr. Protoc. Cell Biol. 55:8.8.1‐8.8.34. © 2012 by John Wiley & Sons, Inc.

The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to a great extent, unknown. Previous studies in vitro showed that nitric oxide (NO) works as a negative chemotropic cue for pollen tube growth in lily (Lilium longiflorum). Furthermore, Arabidopsis thaliana Atnos1 mutant plants, which show defective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen-pistil communication, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnos1 plants shows that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF-2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the micropyle. The pollen-pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1 and two Nitrate Reductases (nr1 and nr2), which collectively are thought to trigger a putative NO signaling pathway. Semi-vivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca(2+) signaling. A role of NO in modulating Ca(2+) signaling was further substantiated by direct imaging the cytosolic free Ca(2+) concentration during NO-induced re-orientation, where two peaks of Ca(2+) occur-one during the slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide evidence for the participation of NO signaling events during pollen-pistil interaction. Of special relevance, NO seems to directly affect the targeting of pollen tubes to the ovule's micropyle by modulating the action of its diffusible factors.

Along the past recent years, two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. The growing body of literature covered in this review points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results covered in this review point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms. Since prices make it one of the most expensive microscopies, TPE is coming slow to be a generalised technology, but enough data are emerging to establish it as a method with no alternative for some objectives.

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