Our data ascribe a biological function for CNP in mediating MT and F-actin reorganization and promoting MT assembly for process outgrowth in OLs. Numerous cytoskeleton-interacting proteins are likely involved in modulating MTs and F-actin along with membrane expansion during myelination. To our knowledge, CNP is the first cytoskeleton effector protein to be described, aside from MAPs, that mediates process outgrowth in OLs.
MAPs bind to MTs to stabilize them against depolymerization (Maccioni and Cambiazo, 1995
). Although OLs express MAP2c and τ abundantly, their functional roles remain unclear (Richter-Landsberg, 2001
). It was recently shown that Fyn kinase and τ interactions promote process outgrowth in OLs (Klein et al., 2002
), and MAP2c is essential for dendritic outgrowth in neurons (Teng et al., 2001
; Harada et al., 2002
). These findings indicate a general role for both MAPs in branch formation by stabilizing assembling MTs. In contrast, CNP binds preferentially to tubulin heterodimers and copolymerizes with tubulin. Thus, a primary function of CNP is in MT assembly rather than in stabilization, which is in contrast to conventional glial MAPs. Interestingly, CNP is functionally related to two neuronal proteins, CRMP2 (Fukata et al., 2002
) and cypin (Akum et al., 2004
), both of which bind tubulin dimers and promote assembly in vitro. In vivo, CRMP2 promotes axonal growth and branching, whereas cypin regulates dendrite branching, suggesting that process extension and arborization can be promoted alternatively by tubulin-copolymerizing proteins.
Using COS-7 cells as a model to study cytoskeletal effects (as a result of their large, flat morphology), we examined how CNP transduces changes to the cytoskeleton to promote process outgrowth that is very much similar to OLs. At early stages of morphogenesis, CNP induces MT destabilization, which is necessary for MT reorganization. This is marked by the visible loss of acetylated MTs, which signals increased MT dynamics in the cell. CNP is enriched in the perinuclear region, which comprises the MTOC and is where frequent MT loss is observed. We propose that CNP indirectly disrupts the preexisting MT array by depleting tubulin subunit pools through its interactions with tubulin. In addition, CNP/tubulin varicosities are present and are aligned along radially extended MTs. These varicosities might be CNP/tubulin oligomers that are peripherally transported to cell edges, where MT assembly occurs. Consistent with previous studies, we also observed detergent-insoluble CNP/tubulin varicosities in association with stable MTs in OL processes (Pereyra et al., 1988
; Gillespie et al., 1989
; Wilson and Brophy, 1989
). Whether tubulin is transported exclusively in subunit form or as a stable polymer remains controversial (Baas, 2002
; Shah and Cleveland, 2002
; Terada, 2003
). Interestingly, oligomeric tubulin complexes are transported by kinesin in giant squid axons (Terada et al., 2000
). In the absence of pharmacological and live cell imaging studies, we can only speculate that these might be transport elements required for MT assembly. Alternatively, these oligomers may serve as nucleation sites for MT assembly (Caudron et al., 2002
). At later stages of morphogenesis, reorganized MTs are stabilized and are marked by the abundant presence of acetylated MTs. Thick, bundled strands are radially projected into the cell periphery, where frayed MT ends extend into filopodia and lamellipodia. As main processes become established, continued MT growth in the processes drives branching. Cytoplasmic extrusions along the processes contain dynamic MTs and are sites for branching, where splayed MTs advance into newly projected F-actin branches. We show that CNP likely promotes MT growth and process arborization by polymerizing MTs at their plus ends. In support of this, process outgrowth is either reduced or abolished in OLs from CNP-null mice as well as in COS-7 and OLN-93 cells overexpressing CNP mutants that are inactive for tubulin polymerization.
CNP also induces F-actin reorganization that is essential for establishing arborized processes by promoting filopodia and lamellipodia formation (De Angelis and Braun, 1994
) and causing cortical actin and stress fiber disassembly. F-actin–driven protrusions, in the form of filopodia and lamellipodia, are indispensable for MT growth in neurites and growth cones (Luo, 2002
) as well as in developing OL processes (Song et al., 2001a
). In contrast, cortical actin can impede MT-driven membrane protusion because it imparts tensile strength to the plasma membrane (Baorto et al., 1992
; Edson et al., 1993
; Lafont et al., 1993
). Stress fibers can also oppose lamellipodia and filopodia formation because cell contractility generally inhibits membrane protrusive activity. Unlike COS-7 cells, both of these F-actin arrays are sufficiently present in HeLa S3 cells to prevent CNP-induced process formation; process extension becomes permissible only upon F-actin disruption using cytochalasin.
CNP's role in process outgrowth appears to be for exclusively myelinating cells, as it is highly expressed only in OLs and Schwann cells. Premyelinating OLs in CNP-overexpressing mice differentiate earlier and produce extraneous membranous extensions at myelin internodes (Gravel et al., 1996
; Yin et al., 1997
). This gain-of-function phenotype is clearly recapitulated by cultured OLs from adult animals exhibiting faster regrowth of dramatically larger and more complex processes (Gravel et al., 1996
). In contrast, CNP-null mice appear to develop normally, showing no apparent myelin abnormalities until 3 mo of age, when paranodal and nodal regions become progressively disorganized (Rasband et al., 2005
). With time, these aberrations lead to axonal degeneration (as early as 5 mo) before outward neurodegenerative symptoms develop, eventually leading to premature death (Lappe-Siefke et al., 2003
). It is believed that axonal death is directly attributed to improper axon–glial cell signaling and organization that is brought on by the failure to form proper axon–glial contacts at the paranodes. This is highly conceivable in the face of numerous data showing axonal loss as a direct consequence of paranode disorganization (Salzer, 2003
How does CNP deficiency result in structural defects to the paranodal loops? CNP is an abundant myelin protein, comprising 4% of total CNS myelin proteins by weight. It is widely distributed throughout the OL cell body, processes, and particularly noncompact myelin compartments such as paranodal loops. In ascertaining whether CNP is required for process outgrowth in OLs, we directly assessed the ability of cultured OLs from 3-mo-old CNP-null mice to regrow their processes at an age when paranodal defects first appear. Morphometric and surface area analysis of the processes revealed a significant reduction in process outgrowth and branching, suggesting that loss of CNP affects the MT cytoskeleton, thus causing structural defects to the processes and paranodal loops. The underlying cytoskeleton forms the structural scaffold to support the architecture of the processes and myelin sheath. Like the myelin sheath, the cytoskeleton is continually maintained by protein turnover and synthesis. CNP is likely to be essential for maintaining proper MT organization in the processes and noncompact myelin compartments, possibly by regulating MT assembly and dynamics, transporting tubulin subunits to these sites, and/or stabilizing MTs directly. On the other hand, an aberrant MT cytoskeleton in CNP-null OLs could effectively impede protein transport, affect the assembly of signaling complexes, and/or alter the structure of paranodal loops and other noncompact myelin compartments; all of this could impair axon–glial signaling. For example, the accumulation of dysfunctional MTs in the taiep myelin mutant rat impedes transport of myelin components, leading to the inadequate maintenance of the myelin sheath (Song et al., 2001b
Similar to the outgrowth of axons and dendrites in neurons, many proteins are likely to contribute to the overall mechanism of process outgrowth in OLs. Although it is clear that CNP is critical for the long-term maintenance of paranodes, additional experiments are planned to address its role in early development during myelination. We reported previously that cultured OL precursors from mutant mice did not exhibit any apparent morphological abnormalities (Lappe-Siefke et al., 2003
); however, unlike our present studies, our earlier assessments lacked quantitative analysis of specific morphological criteria and, thus, may not have detected morphological defects. Also, ultrastructural imaging of the processes and of the MTs within them are warranted. For example, ablation of MAP2, which is essential for dendrite elongation, results in decreased MT density in dendrites (Harada et al., 2002
). Finally, it is also conceivable that alternative mechanisms involving other tubulin-interacting/MT assembly proteins may functionally compensate for CNP deficiency during development, as was shown for τ (Takei et al., 2000
). One possible candidate that might offset CNP loss is CRMP2, which, interestingly, is expressed in OLs in developing and adult brains (Ricard et al., 2000
; Taylor et al., 2004
). If such compensatory mechanisms exist during development, these might be absent in adult OLs, thereby leading to abnormal MT organization and to the development of paranodal defects.