Following fertilisation, the male and female genomes come to occupy a common nuclear compartment, the zygote nucleus 
. This is accomplished either by fusion of the two pronuclear membranes prior to mitosis or mixture of the parental chromosomes following disassembly of the male and female pronuclear envelopes at prophase and reformation of a common nuclear envelope (NE) after mitosis. These strategies represent respectively the so-called “sea urchin type” and “Ascaris
type” distinguished by Wilson 
. In either case, the sperm NE is broken down early in the first cell cycle and a new NE encloses the male genome defining the male pronuclear compartment.
In the sea urchin, once a sperm has entered the egg (which has already completed meiosis) it undergoes a number of transformations before male pronuclear fusion with the female pronucleus 
. Initially, the sperm mitochondrion and flagellum are lost. Formation of the male pronucleus (MPN) is preceded by vesiculation of the sperm NE which is incapable of typical nucleo-cytoplasmic interactions due to lack of nuclear pores. During vesiculation, sperm chromatin decondenses and a new NE is assembled from membranes largely but not completely derived from the endoplasmic reticulum (ER) of the egg 
. New pores also assemble. Migration of the MPN and fusion of its outer and inner nuclear membranes with the female pronucleus result in a zygote nucleus.
This process has been detailed morphologically in intact cells by electron microscopy 
. Extensive biochemical studies on the modification and exchange of sperm and zygotic histone subtypes have been performed on isolated pronuclei 
. However, elucidation of the biochemical details of MPN envelope reformation has depended on development of a cell-free assay 
. In this assay, demembranated sperm nuclei are mixed with fertilised egg extracts and, in the presence of the appropriate nucleotides, complete and functional male pronuclear envelopes are formed. While retaining many features of in vivo
MPN formation, this assay provides a number of experimental advantages: 1) availability of large quantities of synchronous material, making it suitable for analysis with techniques of low read-out sensitivity, 2) ability to manipulate reactions with inhibitors, recombinant proteins and antibodies, and 3) parallel and complementary analyses by traditional biochemical, cell biological and analytical methodologies. Observations from the cell-free assay can be correlated in live sea urchin embryos, which are synchronous and highly suitable for microinjection and microscopy due to their size and transparency 
Our laboratories have described a number of novel aspects of nuclear membrane formation using this assay 
. These include roles for several membrane populations in forming the new NE including sperm nuclear envelope remnants (NERs) and various egg cytoplasmic membranes 
. The material provided by the egg includes ER and Golgi membranes, but more strikingly, an additional population of vesicles, termed MV1 
. MV1 is highly enriched in phospholipase Cγ (PLCγ) and phosphoinositides, including the PLCγ substrate PtdIns(4,5)P2
, when compared to the ER membranes that contribute most of the NE 
. MV1 binds only to the sperm nucleus at the specific polar NER sites, where the remnants of the sperm NE are retained in the acrosomal and centriolar fossae both in vivo
and in vitro
. Addition of GTP leads to the transient phosphorylation of PLCγ, a pre-requisite for its activation 
. Once activated, PLCγ hydrolysis of PtdIns(4,5)P2
forms the fusogenic lipid DAG. The localised accumulation of DAG in MV1 leads to NE formation 
. Initiation of membrane fusion occurs at these sites and subsequently propagates through the ER membranes over the surface of the chromatin to form the fully functional continuous bilayers of the NE 
The signalling events leading to the activation of PLCγ during NE formation are yet to be elucidated. The PLCγ activation process itself is not fully understood. However it is accepted to involve phosphorylation events, including that of Y783 
. More recent findings have enhanced our understanding of the activation process, detailing the auto-inhibitory properties of the PLCγ XY linker region 
, which is mainly mediated by the C-terminus of the two SH2 (cSH2) domains present in the XY linker region. The cSH2 domain was also shown to interact with the Y783 residue when phosphorylated 
. Thus, phosphorylation of Y783 and its subsequent binding to the cSH2 domain is proposed to drive the PLCγ molecule from a closed inactive to an open active state.
In a variety of eukaryotic cells, Src kinases are responsible for the Y783 phosphorylation event 
, and a direct association between a sea urchin Src family kinase (SFK1) and PLCγ has been detected in vitro
. Src kinases are themselves regulated by phosphorylation; they have an inhibitory site at Y527 and an auto-activation site at Y416 
. The C-terminal Y527 site is phosphorylated by Csk 
, rendering the kinase in an auto-inhibited conformation. Dephosphorylation of Y527 followed by the auto-phosphorylation of Y416 leads to Src activation. Thus, though Src kinases are not traditionally thought to be regulators of membrane fusion, they could nonetheless provide a mechanistic link between GTP hydrolysis and PLCγ activation during NE assembly 
. Therefore, we hypothesised that during NE formation, an SFK is responsible for phosphorylating PLCγ on its Y783 residue to achieve full PLCγ catalytic activity 
. To test this we used time resolved Förster resonance energy transfer (FRET) measured by single and two-photon fluorescence lifetime imaging microscopy (FLIM) to examine the proposed SFK-PLCγ interaction in vivo
and in vitro
, and pharmacological studies to assess the SFK-dependency of NE assembly.
Our findings show the direct interaction and temporal regulation of PLCγ and SFK1 in vivo by time-resolved FRET. We also demonstrate that as a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. The ensemble of our data show the phosphorylation and activation of PLCγ by SFK1 during NE assembly both in vivo and in vitro.