GD3 x Tubulin

Primary cilium is a specialized sensory organelle that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials such as tubulin subunits essential for the growing cilia. Here, we show the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis. As the epithelia become polarized, there are more galectin 8 being apically secreted and these extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia through interacting with lipid raft components, such as GD3 ganglioside and scaffold protein caveolin 1. Furthermore, the binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of the transition zone (TZ). Our study also demonstrates the functionality of this barrier depends on intact organization of lipid rafts at the cilia as genetically knockout of Cav1 and pharmacologically inhibition of lipid raft both phenocopy the effect of apical addition of recombinant galectin 8; that is, rapid elongation of primary cilia and redistribution of cilia proteins from TZ to the growing axoneme. Indeed, as cilia elongated, endogenous galectin 8, caveolin 1, and TZ component, TMEM231, also transited from the TZ to the growing axoneme. We also noted that the interaction between caveolin 1 and TMEM231 could be perturbed by exogenous galectin 8. Taken together, we proposed that galectin 8 promoted primary cilia elongation through impeding the barrier function of the TZ by interfering with the interaction between caveolin 1 and TMEM231.

GM3 synthase (ST3GAL5) is the first biosynthetic enzyme of a- and b-series gangliosides. Patients with GM3 synthase deficiency suffer severe neurological disability and deafness. Eight children (ages 4.1 ± 2.3 years) homozygous for ST3GAL5 c.694C>T had no detectable GM3 (a-series) or GD3 (b-series) in plasma. Their auditory function was characterized by the absence of middle ear muscle reflexes, distortion product otoacoustic emissions and cochlear microphonics, as well as abnormal auditory brainstem responses and cortical auditory-evoked potentials. In St3gal5(-/-) mice, stereocilia of outer hair cells showed signs of degeneration as early as postnatal Day 3 (P3); thereafter, blebs devoid of actin or tubulin appeared at the region of vestigial kinocilia, suggesting impaired vesicular trafficking. Stereocilia of St3gal5(-/-) inner hair cells were fused by P17, and protein tyrosine phosphatase receptor Q, normally linked to myosin VI at the tapered base of stereocilia, was maldistributed along the cell membrane. B4galnt1(-/-) (GM2 synthase-deficient) mice expressing only GM3 and GD3 gangliosides had normal auditory structure and function. Thus, GM3-dependent membrane microdomains might be essential for the proper organization and maintenance of stereocilia in auditory hair cells.

In a previous investigation, we demonstrated that after CD95/Fas triggering, raft-associated GD3 ganglioside, normally localized at the plasma membrane of T cells, can be detected in mitochondria, where they contribute to apoptogenic events. Here, we show the association of the glycosphingolipid GD3 with microtubular cytoskeleton at very early time points following Fas ligation. This was assessed by different methodological approaches, including fluorescence resonance energy transfer, immunoelectron microscopy, and coimmunoprecipitation. Furthermore, docking analysis also showed that GD3 has a high affinity for the pore formed by 4 tubulin heterodimers (type I pore), thus suggesting a possible direct interaction between tubulin and GD3. Finally, time-course analyses indicated that the relocalization of GD3 to the mitochondria was time related with the alterations of the mitochondrial membrane potential. Hence, microtubules could act as tracks for ganglioside redistribution following apoptotic stimulation, possibly contributing to the mitochondrial alterations leading to cell death.