Microtubule Depolymerization Affects Endocytosis and Exocytosis in the Tip and Influences Endosome Movement in Tobacco Pollen Tubes

Abstract

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell membrane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching (FRAP) experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.

Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes

“…4). The small area of NtEXO70A1a localization corresponds to the previously described area of exocytosis maximum in the pollen tube (Bove et al, 2008;Idilli et al, 2013;Luo et al, 2016). This localization is in accordance with previous characterizations of Arabidopsis EXO70A1 as the isoform generally responsible for exocytosis in many plant tissues (Synek et al, 2006;Hála et al, 2008;Samuel et al, 2009;Kulich et al, 2010;Drdová et al, 2013;Fendrych et al, 2013;Zhang et al, 2016;Vukašinovi c et al, 2017).…”

“…Alternatively, there can be two subpopulations of NtSEC3a-containing exocyst particles: one binding NtEXO70A1a and the other binding NtEXO70B1. This would also explain why the previously described membrane signal of NtSEC3a (Bloch et al, 2016) seems rather broad in comparison with the narrow area of bulk exocytosis in the tobacco pollen tube (Bove et al, 2008;Idilli et al, 2013;Luo et al, 2016). Second, NtEXO70B1 also could play a role in pollen tube endocytosis.…”

“…Indeed, some PM domains are larger and better separated from each other in this cell type (Sekereš et al, 2015). Zones of active endocytosis (Derksen et al, 1995;Moscatelli et al, 2007) and exocytosis (Bove et al, 2008;Idilli et al, 2013;Luo et al, 2016) are very well separated. A previous study attempting to systematically examine the localization of exocyst components in tobacco (Nicotiana tabacum) BY-2 cells (Chong et al, 2010) resulted in the aggregation of tagged proteins into clusters caused by overexpression in many cases.…”

Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth

“…Conversely, based on colabeling with FM1-43 and FM4-64, it was concluded that exocytosis takes place in a subapical collar located in the transition zone between the tip and the shank, as well as at the shank, but not at the tip (Bove et al, 2008;Zonia and Munnik, 2008). In agreement, the pollen tube-specific syntaxin GFP-SYP124 was observed in the inverted cone, 10 to 25 mm away from the tip (Silva et al, 2010), and fluorescence recovery after photobleaching experiments with FM dyes also have indicated that exocytosis takes place at the subapical region (Bove et al, 2008;Moscatelli et al, 2012;Idilli et al, 2013). Yet, based on pollen tube reorientation experiments in a microfluidics device, it was concluded that growth takes place at the tip rather than at a subapical collar located in the transition zone between the apex and the shank (Sanati Nezhad et al, 2014).…”

Kinesins Have a Dual Function in Organizing Microtubules during Both Tip Growth and Cytokinesis in Physcomitrella patens

“…In tip-growing cells, MTs reside on the cell cortex in a generally longitudinal orientation and contribute to tip growth. In many angiosperm pollen tubes, disruption of the MTs has no apparent effect on growth (Cai and Cresti, 2010), but it has recently been reported to affect both endocytosis and exocytosis in the tip of tobacco (Nicotiana tabacum) pollen tubes (Idilli et al, 2013). The growth of gymnosperm pollen tubes is inhibited by MT-depolymerizing drugs (Anderhag et al, 2000;Justus et al, 2004).…”