Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Structural alterations of spiny stellate cells in the somatosensory cortex in ephrin-A5-deficient mice.

Previous studies demonstrated that in ephrin-A5-deficient mice corticothalamic arbors are reduced by more than 50% in layer 4 of the somatosensory cortex (S1), where ephrin-A5 is normally expressed. Here we examined possible consequences of the reduced thalamic input on spiny stellate cells, the target neurons of thalamocortical afferents. Using ballistic delivery of particles coated with lipophilic dyes in fixed slices and confocal laser-microscopy, we could quantitatively analyze the morphology of these neurons. Cells were examined in S1 at postnatal day 8 (P8), when thalamic afferents establish synaptic contacts and the dendrites of their target cells are covered with filopodia, and at P23, after synapse formation and replacement of filopodia by spines. Our results indicate that at P8 the dendrites of cells in mutant animals exhibit more filopodia and are more branched than dendrites of wildtype cells. In contrast, there is no difference in the extent of the dendritic tree between knockout and control animals. At P23, dendrites of neurons in ephrin-A5-deficient mice are still more branched, but possess fewer spines than wildtype cells. Thus, at early stages layer 4 neurons appear to compensate the reduced thalamic input by increasing dendritic branching and the density of filopodia. However, while at later stages the dendrites of layer 4 neurons in mutants are still more branched, their spine density is now lower than in wildtype cells. Taken together, these data demonstrate that the structure of spiny stellate cells is shaped by thalamic input and Eph receptor signaling.

Activity-regulated dynamic behavior of early dendritic protrusions: evidence for different types of dendritic filopodia.

Dendritic filopodia are long and thin protrusions that occur predominantly during early development of the mammalian CNS. The function of dendritic filopodia is unknown, but they could serve to form early synapses, to generate spines, or to regulate dendritic branching and growth. We used two-photon imaging to characterize the motile behavior of dendritic protrusions during early postnatal development (P2-P12) in pyramidal neurons from acute slices of mouse neocortex. Dendritic protrusions in immature neurons are highly dynamic, and this motility is actin based. Motility and turnover of these early protrusions decreases throughout development, mirroring an increase in their average lifetime and density. Interestingly, density, motility, and length of filopodia are greater in dendritic growth cones than in dendritic shafts. These growth cones disappear after P5. Blocking synaptic transmission globally using TTX or calcium-free solutions led to a 40-120% increase in the density and length of dendritic filopodia in shafts but not in growth cones. Moreover, blocking ionotropic glutamate receptors resulted in an approximately 35% decrease in the density and turnover of shaft filopodia, whereas focal glutamate application led to a 75% increase in the length of shaft filopodia, but neither manipulation affected growth cone filopodia. Our results support the existence of two populations of filopodia, in growth cones and shafts, which are differentially regulated by neuronal activity. We propose that filopodia in dendritic growth cones are involved in dendritic growth and branching in an activity-independent manner, whereas shaft filopodia are responsible for activity-dependent synaptogenesis and, in some cases, may become dendritic spines.

Drebrin-dependent actin clustering in dendritic filopodia governs synaptic targeting of postsynaptic density-95 and dendritic spine morphogenesis.

Dendritic spines have two major structural elements: postsynaptic densities (PSDs) and actin cytoskeletons. PSD proteins are proposed to regulate spine morphogenesis. However, other molecular mechanisms should govern spine morphogenesis, because the initiation of spine morphogenesis precedes the synaptic clustering of these proteins. Here, we show that synaptic clustering of drebrin, an actin-binding protein highly enriched in dendritic spines, governs spine morphogenesis. We immunocytochemically analyzed developing hippocampal neurons of low-density cultures. Filopodia-like dendritic protrusions were classified into two types: diffuse-type filopodia, which have diffuse distribution of drebrin, and cluster-type filopodia, which have drebrin clusters with filamentous actin (F-actin). Most cluster-type filopodia were synaptic filopodia. Postsynaptic drebrin clusters were found in both most synaptic filopodia and spines. Postsynaptic PSD-95 clusters, however, were found in only one-half of synaptic filopodia but in most spines. These data indicate that cluster-type filopodia are not mature spines but their precursors. Suppression of the upregulation of drebrin adult isoform (drebrin A) by antisense oligonucleotides against it attenuated synaptic clustering of PSD-95, as well as clustering of drebrin and F-actin. Furthermore, the restoration of drebrin A expression by injection of the expression vectors of drebrin A tagged with green fluorescent protein into the neurons treated with the antisense oligonucleotides induced synaptic reclustering of PSD-95 on clusters of the labeled drebrin A. These data indicated that the synaptic clustering of drebrin is necessary for that of PSD-95 in developing neurons. Together, these data suggest that synaptic clustering of drebrin is an essential step for spine morphogenesis.

Three functionally distinct adhesions in filopodia: shaft adhesions control lamellar extension.

In this study, adhesions on individual filopodial shafts were shown to control veil (lamellar) advance and to be modulated by guidance cues. Adhesions were detected in individual filopodia of sensory growth cones using optical recordings, adhesion markers, and electron microscopy. Veils readily advanced along filopodia lacking shaft adhesions but rarely advanced along filopodia displaying shaft adhesions. Experiments altering adhesion showed that this relationship is not caused by veils removing adhesions as they advanced. Reducing adhesion with antibodies decreased the proportion of filopodia with shaft adhesions and coordinately increased veil advance. Moreover, the inhibitory relationship was maintained: veils still failed to advance on individual filopodia that retained shaft adhesions. These results support the idea that shaft adhesions inhibit veil advance. Of particular interest, guidance cues can act by altering shaft adhesions. When a cellular cue was contacted by a filopodial tip, veil extension and shaft adhesions altered in concert. Contact with a Schwann cell induced veil advance and inhibited shaft adhesions. In contrast, contact with a posterior sclerotome cell prohibited veil advance and promoted shaft adhesions. These results show that veil advance is controlled by shaft adhesions and that guidance signal cascades can alter veil advance by altering these adhesions. Shaft adhesions thus differ functionally from two other adhesions identified on individual filopodia. Tip adhesions suffice to signal. Basal adhesions do not influence veil advance but are critical to filopodial initiation and dynamics. Individual growth cone filopodia thus develop three functionally distinct adhesions that are vital for both motility and navigation.