LINC complexes are evolutionarily conserved nuclear envelope bridges, physically connecting the nucleus to the peripheral cytoskeleton. They are pivotal for dynamic cellular and developmental processes, like nuclear migration, anchoring and positioning, meiotic chromosome movements and maintenance of cell polarity and nuclear shape. Active nuclear reshaping is a hallmark of mammalian sperm development and, by transducing cytoskeletal forces to the nuclear envelope, LINC complexes could be vital for sperm head formation as well. We here analyzed in detail the behavior and function of Sun4, a bona fide testis-specific LINC component. We demonstrate that Sun4 is solely expressed in spermatids and there localizes to the posterior nuclear envelope, likely interacting with Sun3/Nesprin1 LINC components. Our study revealed that Sun4 deficiency severely impacts the nucleocytoplasmic junction, leads to mislocalization of other LINC components and interferes with the formation of the microtubule manchette, which finally culminates in a globozoospermia-like phenotype. Together, our study provides direct evidence for a critical role of LINC complexes in mammalian sperm head formation and male fertility.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been linked to neuronal plasticity associated with long-term potentiation as well as structural synaptic plasticity. Previous work in adult honeybees has shown that a single CaMKII gene is strongly expressed in the mushroom bodies (MBs), brain centers associated with sensory integration, and learning and memory formation. To study a potential role of CaMKII in synaptic plasticity, the cellular and subcellular distribution of activated (phosphorylated) pCaMKII protein was investigated at various life stages of the honeybee using immunocytochemistry, confocal microscopy, and western blot analyses. Whereas at pupal stages 3-4 most parts of the brain showed high levels of pCaMKII immunoreactivity, the protein was predominantly concentrated in the MBs in the adult brain. The results show that pCaMKII is present in a specific subpopulation of Kenyon cells, the noncompact cells. Within the olfactory (lip) and visual (collar) subregion of the MB calyx neuropil pCaMKII was colocalized with f-actin in postsynaptic compartments of microglomeruli, indicating that it is enriched in Kenyon cell dendritic spines. This suggests a potential role of CaMKII in Kenyon cell dendritic plasticity. Interestingly, pCaMKII protein was absent in two other types of Kenyon cells, the inner compact cells associated with the multimodal basal ring and the outer compact cells. During adult behavioral maturation from nurse bees to foragers, pCaMKII distribution remained essentially similar at the qualitative level, suggesting a potential role in dendritic plasticity of Kenyon cells throughout the entire life span of a worker bee.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (linkers of the nucleoskeleton and the cytoskeleton). Previous studies have demonstrated that the testis-specific SUN domain protein SUN4 is a vital player in the directed shaping of the spermatid nucleus. However, its molecular properties relating to this crucial function have remained largely unknown and controversial data for the organization and orientation of SUN4 within the spermatid nuclear envelope have been presented so far. Here we have re-evaluated this issue in detail and show robust evidence that SUN4 is integral to the inner nuclear membrane, sharing a classical SUN domain protein topology. The C-terminal SUN domain of SUN4 localizes to the perinuclear space, while the N-terminus is directed to the nucleoplasm, interacting with the spermiogenesis-specific lamin B3. We found that SUN4 forms heteromeric assemblies with SUN3 in vivo and regulates SUN3 expression. Together, our results contribute to a better understanding of the specific function of SUN4 at the spermatid nucleo-cytoplasmic junction and the process of sperm-head formation.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (linkers of the nucleoskeleton and the cytoskeleton). Previous studies have demonstrated that the testis-specific SUN domain protein Sun4 is a vital player in spermatogenesis, critically involved in the directed shaping of the spermatid nucleus. Its molecular properties relating to this crucial function, however, have remained largely unknown. Previous studies presented quite controversial data for the general organization and orientation of Sun4 within the spermatid nuclear envelope. In the present study, we have re-evaluated this issue in detail and present new robust data on the Sun4 topology and its interactions at the nucleo-cytoplasmic junction. We identified Sun4 as an integral protein of the inner nuclear membrane, sharing a classical SUN domain protein topology. Similar to other SUN domain proteins, the C-terminal SUN domain of Sun4 locates to the perinuclear space and the N-terminus is directed to the nucleoplasm, where it interacts with the spermiogenesis specific Lamin B3. We found that Sun4 in its natural environment forms heteromeric assemblies with Sun3 and, beyond this, it is crucially involved in the regulation of Sun3 expression. Together, our results contribute to a better understanding of the specific function of Sun4 at the spermatid nucleo-cytoplasmic junction and the entire process of sperm-head formation.Summary statementIn our current study, we have analyzed in detail the biochemical and dynamic properties of the testis-specific SUN domain protein Sun4 and we provide novel insights into its interaction behavior at the spermatid nucleo-cytoplasmic junction.