Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons.Most neurons develop polarity by forming a single long axon and multiple short dendrites (1, 2). The ability of neurons to develop and maintain polarity is essential for their basic functions. The polarization processes have been extensively studied in cultured hippocampal neurons (1, 3). These cells first form several minor processes, any of which appears capable of becoming either an axon or a dendrite, during the first 12-24 h after plating (stage 2). One of the neurites then rapidly elongates to acquire axonal characteristics (stage 3), whereas the others later become dendrites (stage 4). Hippocampal neurons must use a robust internal mechanism that ensures polarization as they regenerate a single axon and multiple dendrites even when polarity is altered by axonal amputation (4, 5).Recent studies have begun to reveal molecules involved in neuronal polarization (6 -8). Localization, overexpression, and loss of function studies showed that spatially localized intracellular signals of a number of molecules, such as phosphoinositide-3-kinase (PI 3-kinase) 2 (9), phosphatidylinositol (3, 4, 5) triphosphate (10), Akt (11), glycogen synthase kinase-3␤ (11-13), collapsin response mediator protein-2 (CRMP-2) (12, 14), mPar3/mPar6/atypical protein kinase C complex (9, 15), Cdc42 (15, 16), Rap1B (16), STEF/Tiam1 (15, 17), Rac (15), adenomatous polyposis coli (18), JNK (19), and DOCK7 (20) are involved in axon specification for neuronal polarity formation. As downstream events, these pathways are thought to regulate cytoskeletal networks of actin filaments (21) and microtubules (20,22).In contrast to the progress in defining the mechanisms of axon specification, our knowledge of how neurons g...