Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

Hyphal extension in fungi requires a tip-high Ca2+ gradient,which is generated and maintained internally by inositol (1,4,5)-trisphosphate(IP3)-induced Ca2+ release from tip-localized vesicles and subapical Ca2+ sequestration. Using the planar bilayer method we demonstrated the presence of two types of IP3-activated Ca2+ channels in Neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). On sucrose density gradients the low conductance channel co-localized with endoplasmic reticulum and plasma membrane, and the high conductance channel co-localized with vacuolar membranes. We correlated the effect of inhibitors on channel activity with their effect on hyphal growth and Ca2+ gradients. The inhibitor of IP3-induced Ca2+ release, 2-aminoethoxidiphenylborate (2-APB), inhibits both channels, while heparin, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate,hydrochloride (TMB-8) and dantrolene inhibit only the large conductance channel. Because 2-APB inhibits hyphal growth and dissipates the tip-high cytosolic [Ca2+] gradient, whereas heparin microinjection, TMB-8 and dantrolene treatments do not affect growth, we suggest that the small conductance channel generates the obligatory tip-high Ca2+ gradient during hyphal growth. Since IP3 production must be catalyzed by tip-localized phospholipase C, we show that a number of phospholipase C inhibitors [neomycin,1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) (but not the inactive pyrrolidine U-73343),3-nitrocoumarin] inhibit hyphal growth and affect, similarly to 2-APB, the location of vesicular Ca2+ imaged by chlortetracycline staining.

A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungus Neurospora crassa. The Ca 2+ gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca 2+ gradients, but this can be explained by Ca 2+ diffusion.Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca 2+ motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP 3 ). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP 3 -activated Ca 2+ channels generate and maintain the tip-high calcium gradient.

Summary. We examined the ionic regulation of tip growth in Neurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from -200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K +) or change dramatically (e.g., Ca > influx) but had no effect on hyphal growth rates. Therefore, ionic fuxes (including Ca > influx) may not be required for tip growth. However, intracellular Ca 2+ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca 2+ directly by ionophoresis. Elevated Ca 2 § induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca 2 § gradient within 10 gm of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca > chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N',N'-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca 2+ and dissipation of the tip-high gradient. We conclude that Ca 2+ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca 2+ gradient is generated from intracellular Ca 2. stores in the ascomycete N. crassa.