The majority of eukaryotic proteins undergo N-terminal (Nt) modifications facilitated by various enzymes. These enzymes, which target the initial amino acid of a polypeptide in a sequence-dependent manner, encompass peptidases, transferases, cysteine oxygenases, and ligases. Nt modifications - such as acetylation, fatty acylations, methylation, arginylation, and oxidation - enhance proteome complexity and regulate protein targeting, stability, and complex formation. Modifications at protein N termini are thereby core components of a large number of biological processes, including cell signaling and motility, autophagy regulation, and plant and animal oxygen sensing. Dysregulation of Nt-modifying enzymes is implicated in several human diseases. In this feature review we provide an overview of the various protein Nt modifications occurring either co- or post-translationally, the enzymes involved, and the biological impact.

3D cell culture assays are becoming increasingly popular due to their higher resemblance to tissue environment. These provide an increased complexity compared to the growth on 2D surface and therefore allow studies of advanced cellular properties such as invasion. We report here on the use of 3D Matrigel cell preparations combined with a particular gentle and informative type of live-cell microscopy: quantitative digital holographic microscopy (DHM), here performed by a commercial software-integrated system, currently mostly used for 2D cell culture preparations. By demonstrating this compatibility, we highlight the possible time-efficient quantitative analysis obtained by using a commercial software-integrated DHM system, also for cells in a more advanced 3D culture environment. Further, we demonstrate two very different examples making use of this advantage by performing quantitative DHM analysis of: (1) wound closure cell monolayer Matrigel invasion assay and (2) Matrigel-trapped single and clumps of suspension cells. For both these, we benefited from the autofocus functionality of digital phase holographic imaging to obtain 3D information for cells migrating in a 3D environment. For the latter, we demonstrate that it is possible to quantitatively measure tumourigenic properties like growth of cell clump (or spheroid) over time, as well as single-cell invasion out of cell clump and into the surrounding extracellular matrix. Overall, our findings highlight several possibilities for 3D digital holographic microscopy applications combined with 3D cell preparations, therein studies of drug response or genetic alterations on invasion capacity as well as on tumour growth and metastasis.

N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization.