Patch-seq reveals new neuronal subtypes by combining electrophysiological and RNA-seq data on single neurons in situ. Despite the importance of the mammalian neocortex for complex cognitive processes, we still lack a comprehensive description of its cellular components. To improve the classification of neuronal cell types and the functional characterization of single neurons, we present Patch-seq, a method that combines whole-cell electrophysiological patch-clamp recordings, single-cell RNA-sequencing and morphological characterization. Following electrophysiological characterization, cell contents are aspirated through the patch-clamp pipette and prepared for RNA-sequencing. Using this approach, we generate electrophysiological and molecular profiles of 58 neocortical cells and show that gene expression patterns can be used to infer the morphological and physiological properties such as axonal arborization and action potential amplitude of individual neurons. Our results shed light on the molecular underpinnings of neuronal diversity and suggest that Patch-seq can facilitate the classification of cell types in the nervous system.

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P₂), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-δ₁, which binds specifically to PtdIns 4, 5-P₂, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P₂ act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P₂ compartmentalization in the regulation of this process.

Rho family GTPases appear to play an important role in the regulation of the actin cytoskeleton, but the mechanism of regulation is unknown. Since phosphoinositide 3-kinase and phosphatidylinositol 4,5-bisphosphate have also been implicated in actin reorganization, we investigated the possibility that Rho family members interact with phosphoinositide kinases. We found that both GTP- and GDP-bound Rac1 associate with phosphatidylinositol-4-phosphate 5-kinase in vitro and in vivo. Phosphoinositide 3-kinase also bound to Rac1 and Cdc42Hs, and these interactions were GTP-dependent. Stimulation of Swiss 3T3 cells with platelet-derived growth factor induced the association of PI 3-kinase with Rac in immunoprecipitates. PI 3-kinase activity was also detected in Cdc42 immunoprecipitates from COS7 cells. These results suggest that phosphoinositide kinases are involved in Rho family signal transduction pathways and raise the possibility that the effects of Rho family members on the actin cytoskeleton are mediated in part by phosphoinositide kinases.